Clustered cell compositions, medical devices therefor, and use in breast reconstruction

Abstract

A kind of cell composition, including the cell cluster consisting of adipose-derived stem cells, endothelial cells and fibroblasts, as fat tissue regeneration unit.It is proved that the cell cluster composition of the application not only promotes the generation of fat, but also makes the generation of fat and angiogenesis and collagen formation etc.parallel, effectively promote the ability of fat tissue regeneration, while solving the problem of survival rate and insufficient vascularization after ADSCs transplantation.The combination of fat tissue regeneration unit and pGelMA not only solves the problem of vascularized fat tissue regeneration, but also promotes the formation of tissue similar to natural mammary tissue, providing a solution for realizing clinical breast reconstruction.

Description

Technical Field

[0001] This invention relates to a biomaterial, and more particularly to a clump-like tissue composed of multiple cells, used to coordinate angiogenesis and extracellular matrix remodeling, and to be used in conjunction with scaffold materials to make medical devices for clinical breast reconstruction. Background Technology

[0002] Autologous fat grafting (AFT) has become an important part of clinical practice in breast enhancement, correction of breast deformities (such as tubular breasts), repair of breast defects after tumor resection, and breast atrophy after lactation. Compared with traditional breast cosmetic and reconstructive surgery (which typically involves implantation or autologous myocutaneous flap transplantation), AFT has advantages such as readily available materials, low immunogenicity, and minimal surgical trauma. However, the low survival rate and calcification of transplanted fat tissue pose challenges to postoperative outcomes, highlighting the need for engineering solutions to improve fat tissue survival.

[0003] Recent studies have shown that adipose-derived stem cells (ADSCs) combined with tissue engineering techniques possess strong adipogenic differentiation potential and demonstrate great promise for breast regeneration. However, the simple combination of ADSCs and biomaterials used in most studies cannot effectively simulate the three-dimensional structure, intercellular and cell-matrix interactions, and mechanical properties in vivo. Three-dimensional cell aggregates offer a strategy to overcome these problems (Pharmacol Ther. 2021; 218:107668). These aggregates are formed through culture in a non-adhesive environment, allowing cells to attach to each other and generate their own extracellular matrix (ECM).

[0004] Adipose tissue is highly vascularized, and the vascularization of newly formed tissue not only provides it with sufficient oxygen and nutrients but also significantly affects its function and homeostasis (Pharmacol Ther. 2022; 231:107976). Therefore, angiogenesis is crucial for the survival of transplanted tissue. However, achieving adequate vascularization in engineered adipose tissue remains a pressing problem. To improve the survival rate and regenerative capacity of engineered adipose tissue, researchers have developed various adipose-generating components with enhanced angiogenesis capabilities, but these components either fail to mimic the complex composition and dynamic characteristics of adipose tissue or are difficult to mass-produce due to time-consuming manufacturing processes. Summary of the Invention

[0005] One object of the present invention is to provide a cell composition comprising a cell cluster of multiple cells for coordinating angiogenesis and extracellular matrix remodeling.

[0006] Another object of the present invention is to provide a cell composition comprising a cell cluster of various cells as an active substance for clinical breast reconstruction.

[0007] Another object of the present invention is to provide the use of a cell composition in the preparation of a medical device for adipose tissue reconstruction.

[0008] Another objective of this invention is to provide a medical device that, when implanted in the body, achieves the clinical purpose of breast reconstruction.

[0009] Collagen fibers, primarily produced by fibroblasts (FBs), are the main component of mammary connective tissue. The structure composed of collagen fibers and other ECM proteins is crucial for the normal performance of biological functions by tissue cells. Therefore, adipogenesis, angiogenesis, and collagen formation are indispensable parts of breast regeneration.

[0010] Studies have found that ADSC spheroids significantly promote adipogenesis and angiogenesis in vivo compared to monolayer ADSCs (Applied Materials Today. 2023; 31:101772). The addition of vascular endothelial cells (VECs) to engineered tissues can promote angiogenesis, thereby promoting tissue regeneration (Biomaterials. 2008; 29:4217–26, Acta Biomother. 2017; 59:317–26).

[0011] The cell composition proposed in this invention is composed of multiple cells and is named: Adipose Tissue Regeneration Unit (ATRU). When implanted into the body, it plays a role in coordinating angiogenesis and extracellular matrix remodeling, thereby achieving the clinical goal of breast reconstruction.

[0012] A cell composition comprising a cluster of adipose-derived stem cells (e.g., adipose-derived mesenchymal stem cells) and endothelial cells as adipose tissue regeneration unit (or micro-tissue, micro-tissue).

[0013] Another cell composition includes a cluster of cells consisting of adipose-derived stem cells and human umbilical vein endothelial cells (HUVECs).

[0014] Another cell composition also includes fibroblasts.

[0015] Another cell composition includes a cluster of cells consisting of adipose-derived mesenchymal stem cells, endothelial cells, and fibroblasts.

[0016] The cell composition of the present invention is in the form of a cell cluster with a volume of approximately 113,100 μm. 3 ~523600μm 3 .

[0017] In the cell composition of the present invention, the ratio of adipose-derived stem cells to fibroblasts is 8:2 to 9:1.

[0018] The cell composition of the present invention has an endothelial cell to fibroblast ratio of 1:1 to 1:1.5.

[0019] The cell composition of the present invention is placed in a porous biodegradable material carrier to obtain engineered microtissues, which are beneficial for microtissues containing clumps of cells to play a role in coordinating angiogenesis and extracellular matrix remodeling, such as promoting the vascularization of grafts.

[0020] Biodegradable materials, such as chitosan, PLA-PEG copolymer, PLGA-PEG copolymer, gelatin, and GelMA, can be formulated into porous gels. These biomaterials facilitate the placement of cell compositions and provide the space needed for cell growth, expansion, and differentiation. For example, a porous GelMA gel can be prepared by preparing an 8wt%–12wt% solution and then cross-linking it under UV light.

[0021] To protect bioengineered microtissues from tensile, compressive, and shear forces that could impair their growth, polylactic acid (PLA) is used as a material to 3D print breast-like aesthetic scaffolds, which serve as molds for breast reconstruction. A solution of porous GelMA gel is mixed with a cell composition, cross-linked under ultraviolet light, and the resulting engineered microtissues are placed into the aesthetic scaffold to create a medical device that is implanted in the body, thereby achieving the clinical goal of breast reconstruction.

[0022] Verification has shown that the clumped cell composition of the present invention not only promotes fat generation, but also enables angiogenesis and collagen formation to occur in parallel with fat generation, effectively promoting the regeneration capacity of adipose tissue, while solving the problems of low survival rate and insufficient vascularization after ADSC transplantation.

[0023] Multi-cell co-cultured clumps of micro-tissues (denoted as s-AF, s-AH, and s-AFH) exhibited greater adipose tissue regeneration compared to single-cell spheroids (sA). The addition of HUVECs significantly enhanced vascularization, as evidenced by increased vessel numbers observed in the s-AH and s-AFH groups at 4 and 8 weeks. The addition of fiber bundles (FBs) promoted type III collagen formation, crucial for breast tissue structural integrity. The combined use of HUVECs and FBs also resulted in an overall increase in collagen fiber production.

[0024] By combining multiple cell types, a more robust and functional microtissue was created, better mimicking the environment of natural breast tissue. Furthermore, the use of GelMA hydrogel provides a highly biocompatible and elastic matrix that facilitates cell growth and tissue integration, while the porous structure of the hydrogel supports cell migration and nutrient exchange. Moreover, the combination of microtissue and pGelMA not only addresses the problem of vascularized adipose tissue regeneration but also promotes tissue formation similar to natural breast tissue.

[0025] Based on the histological characteristics of the target adipose tissue, tissue reconstruction for different anatomical sites can be tailored by adjusting the cell ratio and type in the micro-tissue of the present invention. Attached Figure Description

[0026] Figure 1 A schematic diagram of a process for performing clinical breast reconstruction using the cell composition of the present invention;

[0027] Figure 2 The figures show the characterization results of pGelMA and PLA; where A is a schematic diagram of pGelMA hydrogel, B is a scanning electron microscope (SEM) image of pGelMA hydrogel, C is a schematic diagram of 3D printed PLA scaffold, D is an SEM image of the microstructure of the PLA scaffold surface, E is a schematic diagram of compression test, F is the compression test result of pGelMA hydrogel (showing its excellent elasticity), G is the compression modulus diagram of pGelMA, PLA and their composites, and H is the CCK-8 test result.

[0028] Figure 3 The figures show the results of cell spheroid culture and characterization. Among them, A is a schematic diagram of the cell spheroid culture process, B is an image of cell spheroids in microplates at different time points, C is the cell spheroid disintegration observed on day 3, and D is a scanning electron microscope (SEM) image, which provides detailed structural information of the cell spheroids.

[0029] Figure 4 The figures show the results of cell growth, migration, and adipogenic differentiation. "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, "****" indicates p < 0.0001, and "ns" indicates no significant difference. Specifically, A shows images of cell spheroids on day 1 and day 3 of co-culture in hydrogel using phase-contrast microscopy; B shows images of the same area taken using fluorescence microscopy, demonstrating cell migration behavior; C shows the results of tube formation; D shows the results of Oil Red O staining; and E shows the quantitative results of newly formed junctions and branching lengths in each experimental group after treatment with cell culture medium or PLA-pGelMA extract.

[0030] Figure 5 The images show the results of cell proliferation, adipocyte differentiation, and collagen formation in hydrogels. "*" indicates p < 0.05, "**" indicates p < 0.01, "***" indicates p < 0.001, "****" indicates p < 0.0001, and "ns" indicates no significant difference. Specifically, A shows Ki-67 staining of cell spheroids after 7 days of co-culturing with hydrogels in different groups; B shows double staining of cell spheroids (FABP4, green; PPAR-γ, red) after 7 days of co-culturing with hydrogels in different groups; and C shows representative immunofluorescence staining images of cell spheroids after 7 days of co-culturing with hydrogels in different groups. DAPI (blue), COL-1 (green), and COL-3 (red). D is a quantitative statistical chart of Ki-67 staining in cell spheroids in different groups. E is a quantitative statistical chart of the intensity of double staining of FABP4 and PPAR-γ in cell spheroids in each experimental group. F is a quantitative statistical chart of the positive areas of FABP4 and PPAR-γ in cell spheroids in each experimental group. G is a quantitative statistical chart of the positive area of ​​COL-1 in cell spheroids in each experimental group. H is a quantitative statistical chart of the positive area of ​​COL-3 in cell spheroids in each experimental group. I is a quantitative statistical chart of the ratio of positive area of ​​COL-1 to positive area of ​​COL-3 in cell spheroids in each group.

[0031] Figure 6 The figure shows the results of the in vivo fat regeneration test of cell spheroids. In the figure, "*" indicates p<0.05, "**" indicates p<0.01, and "ns" indicates no significant difference. Among them, A is the perilipin staining map at 2, 4 and 8 weeks, B is the PPAR-γ staining map, C is the quantitative statistical map of fat region of each experimental group at each time point, and D is the quantitative statistical map of PPAR-γ fluorescence intensity of each experimental group at 8 weeks.

[0032] Figure 7 The graphs show the validation of angiogenesis and collagen formation in vivo. In the graphs, "*" indicates p<0.05, "**" indicates p<0.01, "***" indicates p<0.001, "****" indicates p<0.0001, and "ns" indicates no significant difference. Among them, A is the CD31 staining map of each experimental group at 2, 4, and 8 weeks; B is the Masson trichrome staining map of each experimental group at 8 weeks; C is the Sirius Red staining map of each experimental group at 8 weeks; D is the quantitative statistical graph of CD31 staining of each experimental group; E is the quantitative statistical graph of Masson trichrome staining; and F is the quantitative statistical graph of Sirius Red staining of each experimental group. Detailed Implementation

[0033] The technical solution of the present invention will be described in detail below with reference to the accompanying drawings. The embodiments of the present invention are only used to illustrate the technical solution of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solution of the invention without departing from the spirit and scope of the technical solution of the present invention, and all such modifications and substitutions should be covered within the scope of the claims of the present invention.

[0034] This invention utilizes cellular compositions to form engineered microtissues to coordinate angiogenesis and extracellular matrix remodeling. A schematic diagram of clinical breast reconstruction can be found here. Figure 1 The specific experimental methods used are described below:

[0035] 1) Materials

[0036] Microwell culture plates and anti-adhesion cleaning solution were purchased from STEMCELL Technologies. High-glucose DMEM cell culture medium, low-glucose DMEM cell culture medium, and fetal bovine serum (FBS) were all from Gibco (UK). Porous gelMA and ultraviolet light generation equipment were purchased from Suzhou Institute of Intelligent Manufacturing.

[0037] 2) Adipocytes and laboratory animals

[0038] ADSCs isolated from two healthy female donors via liposuction were obtained from Shanghai Ninth People's Hospital and approved by its ethics committee. Human FBs and HUVECs were cell lines purchased from the Stem Cell Bank of the Chinese Academy of Sciences. All male nude mice were obtained from the Laboratory Animal Center of Shanghai Ninth People's Hospital, and all animal experiments complied with the ethical standards of Shanghai Jiao Tong University School of Medicine.

[0039] 3) Preparation and characterization of porous GelMA hydrogels

[0040] Cell-free porous GelMA (pGelMA) hydrogels were prepared by dissolving pGelMA in phosphate-buffered saline (PBS) to form an 8% solution, followed by crosslinking into specific shapes using ultraviolet light (405 nm, 10 s). Cell-containing pGelMA hydrogels were prepared by mixing filtered 8% pGelMA solution with cell spheres and then crosslinking them using ultraviolet light.

[0041] The microstructure of the pGelMA hydrogel was observed using scanning electron microscopy (SEM) after vacuum drying. To test the mechanical properties of pGelMA, it was injected into a cylindrical mold to create a substrate with a surface area of ​​0.8 cm². 2 A cylindrical bracket with a length of 5mm was used, and a compression test was performed using a mechanical testing instrument.

[0042] 4) Preparation and characterization of PLA scaffolds

[0043] The PLA hemispherical scaffold was fabricated using an SLA660 3D printer (ZRapid Tech, Suzhou, China) with an infill ratio of 20%, a nozzle diameter of 0.1 mm, and inner and outer diameters of 0.9 mm and 1 mm, respectively. SEM images of the scaffold's surface morphology were taken in nude mice before and after implantation to evaluate the PLA scaffold's degradation behavior in vivo.

[0044] 5) CCK-8 cytotoxicity test

[0045] The cytotoxicity of pGelMA, PLA, and their complexes to cells was assessed using the CCK-8 assay. 150 μL of pGelMA hydrogel, a PLA scaffold, and their complexes were each immersed in 5 mL of complete culture medium for 7 days to obtain extracts. ADSCs, FBs, and HUVECs were seeded in 96-well plates at densities of 3000, 3000, and 2000 cells / well, respectively. 10 μL of extract and 90 μL of complete culture medium were added to each well, or 100 μL of complete culture medium was added directly. The culture medium or extract was changed every two days during culture, and the absorbance at 450 nm was measured on days 3 and 7.

[0046] 6) Extraction and Differentiation of ADSCs

[0047] ADSCs were extracted according to previously reported methods. In short, the collected adipose tissue was washed with PBS to remove excess red blood cells. The washed adipose tissue was then immersed in an equal volume of PBS containing 3% antibiotic-antifungal solution (S120JV, Biosbio, Shanghai, China) for 10 minutes, followed by digestion with an equal volume of 0.2% (w / v) collagenase I (Nordmark, Germany) for 2 hours in a constant-temperature shaker (37°C, 120 rpm / min). The digested tissue was filtered and centrifuged at 1200 rpm for 5 minutes. The precipitate containing ADSCs was collected and resuspended in low-glucose medium supplemented with 10% FBS and 1% antibiotic-antifungal agent. Cells were expanded to passage 3 for subsequent experiments.

[0048] In the adipogenic differentiation experiment, cell spheres containing ADSCs were mixed with adipogenic differentiation medium ( HUXMD-90031 (Cyagen Biosciences, Guangzhou, China) was cultured for 7 days, and lipid production was detected by Oil Red O staining.

[0049] 7) Culture and characterization of cell spheroids

[0050] Three cell types (ADSC, FB, and HUVEC) were used to culture four types of cell spheroids: ADSC spheroids (sA), ADSC / FB spheroids (s-AF), ADSC / HUVEC spheroids (s-AH), and ADSC / FB / HUVEC spheroids (s-AFH). For cell spheroids co-cultured with two cell types, the cell ratio (x / y) was 9:1, where x represents ADSC and y represents either FB or HUVEC. For ADSC / FB / HUVEC spheroids, the ratio of the three cell types (x / y / z) was 9:0.5:0.5, where y represents FB and z represents HUVEC. These cell ratios were determined not only based on the study's goal of promoting adipogenesis but also considering the feasibility of obtaining these three cell types clinically.

[0051] Cell spheres are produced using AggreWell TM 800 Microwell culture plates (STEMCELL Technologies Germany GmbH, Cologne, Germany) were used for preparation. Each plate contains 24 wells, with approximately 300 microwells per well. The simplified procedure is as follows: Different cell types were collected and resuspended in high-glucose complete medium to prepare a cell suspension with a concentration of 1.0 × 10⁵ cells / mL. 1 mL of the cell suspension was added to each well, and the plate was centrifuged at 100 × g for 3 minutes to ensure approximately 333 cells per microwell. The plates were then incubated at 37°C in a 5% CO₂ incubator, with 50% of the medium replaced every two days.

[0052] The formation and growth of cell spheres were observed and photographed using a phase-contrast microscope (Nikon ECLIPSE E 100, Nikon Corporation, Japan) on days 0, 1, 3, 5, and 7. To characterize the microscopic morphology of the cell spheres, images were taken using a scanning electron microscope (SEM, Regulus 8100, Hitachi Corporation) on day 3.

[0053] 8) Growth and migration of cell spheres in hydrogels

[0054] Hydrogels loaded with cell spheroids (sA, s-AF, s-AH, s-AFH) were injected into 6-well plates, with each well containing 300 μL of hydrogel and approximately 400 cell spheroids. After UV crosslinking of the hydrogel solution, 2 mL of complete culture medium was added to each well. The growth of different cell spheroids was monitored on days 1 and 3 using a phase-contrast microscope (Nikon ECLIPSE E 100, Nikon Corporation, Japan). Changes in cell spheroid size were analyzed using ImageJ software. Furthermore, the migration of cell spheroids within the hydrogel was observed using a fluorescence microscope (Leica DMi8, Germany) under the same field of view, and data were recorded on days 1 and 3.

[0055] 9) Tube forming experiment

[0056] In a 96-well plate, 50 μL of Matrigel at a concentration of 10 mg / mL was added to each well, and the plate was incubated at 37°C with 5% CO2 for 30 minutes until gelation. After gelation, 100 cell spheres were suspended in 100 μL of cell culture medium containing either 1% FBS (control group) or 10% pGelMA-PLA extract, prepared as described in the previous CCK-8 experiment. Subsequently, 100 μL of cell sphere suspension was added to each well, with three replicates per group, and incubation continued for 6 hours. Images were captured using a phase-contrast microscope (Nikon ECLIPSE E 100, Nikon Corporation, Japan), and data were analyzed using ImageJ software.

[0057] 10) Cell spheroid proliferation, adipogenesis, and collagen formation in hydrogels (in vitro experiments)

[0058] Hydrogels loaded with cell spheroids (sA, s-AF, s-AH, s-AFH) were injected into 24-well plates, with each well containing 300 μL of hydrogel and approximately 600 cell spheroids. After UV crosslinking, complete culture medium was added to each well. After 7 days of culture, samples were taken for section staining analysis. Ki-67 staining was used to detect cell spheroid proliferation in the hydrogel. FABP4 and PPAR-γ staining were used to assess adipogenic activity. Collagen I and Collagen III staining were used to detect collagen formation in the cell spheroids. All section staining results were analyzed using ImageJ software.

[0059] 11) Preparation of PLA-pGelMA-cell sphere complex

[0060] PLA scaffolds were sterilized using ethylene oxide, and pGelMA solution was sterilized by filtration through a 0.22 μm filter membrane. 150 μL of pGelMA solution containing approximately 300 cell spheres was added to each PLA scaffold to prepare a PLA-pGelMA-cell sphere complex (PLA-pGel-CS), which was then subjected to UV crosslinking fixation.

[0061] 12) Nude mouse in vivo transplantation experiment

[0062] Thirty-six six-week-old male nude mice were randomly divided into four groups, receiving PLA-pGel-sA, PLA-pGel-s-AF, PLA-pGel-s-AH, and PLA-pGel-s-AFH treatments, respectively. Prior to transplantation, PLA-pGel-CS were cultured in adipose differentiation medium for 7 days. The breast repair model was constructed according to a previous study (Applied Materials Today. 2023; 31:101772). Specifically, after anesthesia, an incision was made in the upper back of the nude mice, and a scaffold was implanted at each symmetrical location. The wounds were then sutured with 5-0 sutures.

[0063] 13) Histological, immunohistochemical and immunofluorescence staining

[0064] All nude mice were sacrificed at 2, 4, and 8 weeks post-surgery, and the grafts were removed. After recording images of the front and back of the grafts, they were fixed in 4% paraformaldehyde. Subsequently, the PLA scaffold in the fixation complex was removed, and the regenerated tissue was embedded in a paraffin block. Sections were prepared and analyzed by hematoxylin-eosin (H&E) staining, immunohistochemistry, and immunofluorescence staining.

[0065] Example 1: Characterization of hydrogel and PLA scaffold

[0066] Given the critical importance of biocompatibility and non-toxicity of hydrogels in tissue regeneration, GelMA hydrogel was selected as the cell carrier in this embodiment. Porous GelMA (pGelMA) hydrogel was used in all subsequent experiments. To verify the microstructure of pGelMA, its porous structure was confirmed by scanning electron microscopy (SEM). Figure 2 B).

[0067] Through compression testing ( Figure 2 F) Evaluate the mechanical properties of pGelMA, PLA, and PLA-pGelMA composites. Photocured pGelMA was formed into cylindrical shapes. Figure 2 A) It remains intact when compressed to 40% of its original height and returns to its original shape after the pressure is removed, demonstrating its excellent elasticity. Figure 2 F). PLA scaffolds designed for shaping hydrogels, providing breast-like aesthetics, and protecting micro-tissues from damage. Figure 2 C) It exhibits strong pressure resistance. Figure 2 G).

[0068] The in vivo degradation behavior of pGelMA hydrogel and PLA scaffold was also tested. Microscopic images of the PLA scaffold surface were captured by SEM 8 weeks before and after implantation in nude mice. Figure 2(D) The results showed that the PLA scaffold had a smooth surface before implantation, but became rough and degraded after 8 weeks, indicating its degradation behavior in vivo. After removal of regenerated tissue, samples were observed at 8 weeks, showing complete degradation of the hydrogel within the scaffold, indicating that the pGelMA hydrogel has high biodegradability.

[0069] To assess the cytotoxicity of cells to different treatments, a CCK-8 assay was performed. ADSCs, FBs, and HUVECs were seeded in 96-well plates and treated with extracts of pGelMA, PLA, and their complexes, respectively. Cell viability was assessed by measuring absorbance at 450 nm on days 3 and 7. Figure 2 The results showed that PLA, pGelMA, and their complexes were not cytotoxic to ADSCs, FBs, and HUVECs. Cell viability was significantly improved on day 7 of ADSC culture. High cell viability indicates good biocompatibility of all materials, supporting their potential for tissue engineering applications.

[0070] Example 2: Culture and Characterization of Clustered Cell Spheroids

[0071] Cell spheroids were cultured using microplates. Figure 3 A). To assess the aggregation behavior of cell spheroids, images of different cell spheroids were captured at different time points using phase-contrast microscopy. Figure 3 B). The images show that cells began to aggregate within two hours, and by 24 hours, most cells in the microplate had aggregated into cell spheroids. The volume of the cell spheroids also increased with prolonged culture time, with s-AH and s-AFH being significantly larger than sA and s-AF. Notably, on day 3, the cell spheroids began to disintegrate. Figure 3 C). However, this disintegration phenomenon was not observed in FB spheres and FB / HUVEC spheres, indicating that the disintegration phenomenon is related to ADSCs.

[0072] Example 3: Growth, migration, and adipogenesis of clump-like cell spheroids in pGelMA

[0073] Cell spheroids were loaded into pGelMA by mixing with pGelMA solution and crosslinking with UV light to evaluate the migration behavior of cell spheroids in porous hydrogels. Images were captured using phase contrast microscopy and fluorescence microscopy on days 1 and 3.

[0074] On the first day of co-culture, the cell spheroids exhibited strong migration ability. By the third day, the migration was even more pronounced, demonstrating the excellent migration ability of the cell spheroids in the porous hydrogel. Figure 4 A).

[0075] Images of the same cell spheroids were captured using a fluorescence microscope, further demonstrating their migration behavior in the hydrogel. Figure 4 (B) We also assessed their growth in the hydrogel by comparing the size of the cell spheroids on day 1 and day 3. The results showed that s-AH and s-AFH had larger volumes and faster growth rates.

[0076] Furthermore, the angiogenic efficacy of the cell spheroids was evaluated using a lumen formation assay. Each group of cell spheroids was treated with either cell culture medium or PLA-pGelMA extract for 6 hours. Representative images were captured and are presented below. Figure 4 C), and in Figure 4 E provides quantitative results for the intersection points and branch lengths.

[0077] Compared with the control group sA, the number of cell spheroids containing HUVECs significantly increased in junction and branch length, and there was no significant difference between the sA and s-AF groups, indicating that HUVECs have strong angiogenesis potential. Cell spheroids treated with PLA-pGelMA extract showed a slight decrease in junction and branch length, but the difference was not statistically significant, indicating that PLA and GelMA hydrogels had limited negative impact on angiogenesis.

[0078] Furthermore, considering that the cell spheroids were co-cultured with adipogenic differentiation medium for 7 days before transplantation into animals, oil red O staining was used to investigate whether lipid droplet formation occurred at this time point. The results showed that in all four groups, a large number of lipid droplets were present around the cell spheroids. Figure 4 D).

[0079] Example 4: Cell spheroid proliferation, adipocyte differentiation, and collagen formation in hydrogel

[0080] To further evaluate the biological behavior of cell spheroids in the hydrogel, including proliferation, adipogenic differentiation, and collagen formation, in vitro immunofluorescence staining was performed.

[0081] The proliferation of cell spheroids was assessed by Ki-67 staining. Figure 5 A). The results showed that the number of Ki-67 positive cells increased in the s-AF, s-AH, and s-AFH groups, with the highest number of Ki-67 positive cells in the s-AH group. Figure 5 D). This observation is attributed to the rapid proliferation rate of HUVECs.

[0082] Adipate differentiation was assessed by double staining FABP4 and PPAR-γ. Figure 5 B). The fluorescence intensity of the staining showed no statistically significant difference among the four groups. Figure 5E). However, compared to single-cell spheroids (sA), co-cultured cell spheroids (s-AF, s-AH, and s-AFH) exhibited larger volume and a wider migration range, resulting in larger positive areas for FABP4 and PPAR-γ, especially in the s-AH and s-AFH groups. Figure 5 F).

[0083] Collagen formation was analyzed by staining type I and type III collagen. Figure 5 C), the merged images showed that the addition of HUVECs may have significantly reduced type III collagen production. Compared with other groups, the s-AF group showed a larger positive area for type I collagen (C). Figure 5 G). Compared with the control group, the s-AFH group also showed an increase in the positive area of ​​type I collagen, but this increase was not statistically significant. Cell spheroids containing HUVECs (s-AH and s-AFH) showed significantly lower type III collagen production than the other two groups, and no positive results were observed in the center of the cell spheroids. Figure 5 H). This finding is consistent with subsequent in vivo experimental results.

[0084] These results indicate that FB binding in spheroids enhances type I collagen production in vitro, leading to an increased ratio of type I to type III collagen in the s-AF group. In contrast, HUVEC binding reduces type III collagen production and significantly increases the ratio of type I to type III collagen in both the s-AH and s-AFH groups. Figure 5 I).

[0085] Example 5: In vivo regeneration of mammary gland-like adipose tissue

[0086] Cell spheroids were embedded in GelMA hydrogel and then injected into PLA to form PLA-pGel-CS constructs to evaluate the regenerative capacity of the cell spheroids in vivo. After culturing in adipocyte differentiation medium for 7 days, the PLA-pGel-CS constructs were implanted into symmetrical sites on the upper back of mice.

[0087] All samples collected from nude mice were stained with perilipin at 2, 4, and 8 weeks post-implantation to assess adipose tissue regeneration. At 2 weeks, adipose tissue comprised approximately 50% of the sections, with no statistically significant difference between groups. Regenerated tissue was primarily located in the center of the sections. By 4 weeks, adipose tissue comprised approximately 60% of the sections, increasing to approximately 75% by 8 weeks. Figure 6 A). Compared with the sA group, co-cultured cell spheroids (s-AF, s-AH, and s-AFH) exhibited faster lipid regeneration, with statistically significant differences observed at 8 weeks. Figure 6 C).

[0088] In addition to perilipin staining, the expression of PPAR-γ, a key regulator of adipogenic differentiation, was also investigated in different groups. Figure 6 B). The results showed that the fluorescence intensity of co-cultured cell spheroids was significantly higher than that of single cell spheroids (B). Figure 6 D), which indicates that the co-cultured cell spheroids have a stronger ability to differentiate into lipids.

[0089] Besides adipogenesis, angiogenesis is also crucial. CD31 immunohistochemical staining was performed on samples collected at 2, 4, and 8 weeks (…). Figure 7 A) was used to assess vascularization in regenerated tissue. At 2 weeks post-implantation, significant evidence of angiogenesis was observed in all groups, with no statistically significant difference in vessel number among the four groups. However, at 4 weeks, the number of vessels in the sA and s-AF groups was reduced compared to 2 weeks. This reduction in vascularization may be attributed to hypoxia-induced angiogenesis in the early stages of regeneration, followed by remodeling and regression of vessels as the tissue stabilized. By week 4, the cell spheroids containing HUVECs showed stronger vascularization capacity than the group without HUVECs. This trend continued, and at 8 weeks, the vascularization of the cell spheroids containing HUVECs (s-AH and s-AFH groups) increased further, significantly exceeding that of the non-HUVEC groups. Furthermore, the vascularization level in the s-AF group was also improved compared to the sA group. These findings suggest that the addition of HUVECs effectively promotes vascularization in regenerated tissue. In addition, co-culture of FBs and ADSCs also promoted vascularization in regenerated adipose tissue. Figure 7 D).

[0090] Similarly, collagen fibers are a major component of breast connective tissue. Therefore, collagen formation in different groups was assessed at 8 weeks using Masson's trichrome staining and Sirius Red staining. Figure 7 B). Masson's trichrome staining results showed that collagen formation was significantly increased in the group containing FBs compared to the group without FBs. Conversely, collagen formation was reduced in the s-AH group compared to the sA group. Figure 7 E). Sirius Red staining analysis showed a similar trend to Masson staining, but the difference was more significant. Figure 7 C and 7F).

Claims

1. A cell composition, characterized in that... It includes cell clusters composed of adipose-derived stem cells and endothelial cells, serving as adipose tissue regeneration units.

2. The cell composition according to claim 1, characterized in that... It also includes fibroblasts.

3. The cell composition according to claim 2, characterized in that... The ratio of adipose-derived stem cells to fibroblasts was 8:2 to 9:

1.

4. The cell composition according to claim 2, characterized in that... The ratio of endothelial cells to fibroblasts was 1:1 to 1:1.

5.

5. The cell composition according to claim 1, characterized in that... The endothelial cells mentioned are human umbilical vein endothelial cells.

6. The cell composition according to claim 1, characterized in that... The cell cluster volume is 113100 μm. 3 ~523600μm 3 .

7. The use of the cell composition according to claim 1 in the preparation of a medical device for adipose tissue reconstruction.

8. The use of the cell composition according to claim 1 in the preparation of a medical device for breast reconstruction.

9. A medical device for breast reconstruction, characterized in that... Includes biodegradable materials and the cell composition of claim 1.

10. The medical device according to claim 9, characterized in that... The biodegradable material is GelMA, which has a porous structure.

Images