This is a retrospective cohort study conducted between December 2015 and December 2022, in which 25 patients with breast cancer who had undergone total mastectomy underwent successful fat-only total breast reconstruction using cultured mature adipocytes and conditioned medium containing MCP-1. This study was approved by the Institutional Review Board of Yanaga Clinic on February, 22, 2015. And then, the study was conducted in accordance with all relevant and applicable governmental and institutional guidelines and regulations, as well as cell therapy guidelines set out by Japanese regenerative medicine law since December, 2015. Yanaga Clinic and Tissue Culture Laboratory, is certified as a Class 2 Regenerative Medicine Cell Processing Facility (registration number: FC7140009; 23 March, 2015) and a Class 2 Regenerative Medicine Provider (registration number for autologous cultured adipocytes transplantation: PB7150005, 11 November, 2015). Clinical outcomes, efficacy, and patient safety were reported as required annually to a government-certified committee on Regenerative Medicine (Gamagori City Hospital Deliberation Committee for Specific Regenerative Medicine as an external committee), the Kyushu office of the Ministry of Health regenerative medicine committee, and finally the Japanese Ministry of Health committee on regenerative medicine. Informed consent was obtained from all participants, and the study protocol conformed to the ethical principles in the Declaration of Helsinki and was approved by the respective institutional review boards.
Preparation of human mature adipocyte culture and condition medium
Approximately 3 cc of fat was harvested from the patient’s lower abdomen by subcutaneous aspiration, washed, sterilized with antibiotics, digested with collagenase, finely filtered with cell strainer, and centrifuged to collect only the fat component floating in the supernatant. Blood components containing ASCs + SVF precipitated as a pellet in the lower layer were discarded. The microfat particles (Fig. 1A) were cultured in a medium consisting of 10% autologous serum added to α-MEM medium.
From 2.8 ± 0.9 ml of aspirated fat collected, 6.92 × 105 ± 3.25 x 105 adipocytes were isolated. The number of cells obtained at P0 passaging was 10.1 × 106 ± 2.8 × 106, of which 1 × 106 was seeded into one 175 cm2 flask and the remaining cells were frozen and stored. The number of cells obtained from P1 passaging was 11.6 × 106 ± 6.2 × 106. Of these, 5 × 105 were seeded per 150 cm2 flask. The remaining cells were cryopreserved. The final number of cells seeded in the 150 cm2 flasks for transplantation was 4.9 × 106 ± 1.6 × 106. (Fig. 1B).The cell count at final transplantation was 17.00 × 107 ± 3.09 × 107. The process from the cell harvest to transplantation was 122.2 ± 100.18 days. In the final phase, after the cryopreserved cells are thawed, they are cultured and transplanted in a period of 13.3 ± 1.2 days.
The fat droplets of cultured cells were stained with Sudan III (Fig. 1C). All cells collected from patients were labeled and will be stored at the cell processing facility at 1.0 × 106 cells/tube for 10 years.
Ability of angiogenesis and adipogenesis in CMAs in vitro
One group of CMAs alone and one group of CMAs plus aspirated fat graft were prepared (n = 6) and cultured for five days to determine whether the CMAs contained PDGFRβ (+) preadipocyte cells and VEGFR2 (+) endothelial progenitor cells. Cells in the wells were fixed in 4% paraformaldehyde. The reagents used were anti-VEGFR2 polyclonal antibody (Bioss, 1: 100) as the primary antibody, anti-PDGFRβ polyclonal antibody [Y92]-C-terminal (Abcam, 1:100), goat anti-rabbit IgG H&L (HRP) (Abcam, 1:200) as the secondary antibody, and DAB substrate kit (Funakoshi, SK-4100, [Vector Laboratories, Inc.]) (Fig. 1D).
Cell surface marker characterization of CMAs
Flow cytometry (Guava easyCyteHT System, easyCyte5HT, S/N: 8470050255, Luminex Corporation) was performed to characterize the cell surface marker profile of human CMA.
Mature adipocytes (n = 6) and SVF cells (n = 6) were isolated and cultured from the same tissue of six individuals. To eliminate individual differences, the mature adipocytes from the six individuals were mixed and cultured in group A and the SVF cells from the same six individuals were cultured in group B. Cells from both groups were detached with trypsin solution and collected. Cells were aliquoted into tubes to make 1 × 106 cells. The cells were immunoassayed with 5 μg antibodies for 30 min at 4 °C listed below, and 10,000 cells were measured in a flow cytometer. Using the measurement results, the expression of MSC markers CD166, CD29, CD105, CD73, CD44, CD90 in both groups were analyzed (Fig. 2).
To calculate CD166, CD29, CD105, CD73, CD44 and CD90 percentage in CMAs and SVFs cells respectively, the analysis was performed as follows (Fig. 2): in total, 10,000 events were recorded by flow cytometry (Guava easyCyte5HT, Luminex Corporation) and the resultant data were analyzed by Guava InCyte software (Luminex Corporation). Events were gated according to their FSC/SSC profile (gate R1) to exclude cellular debris. Expression profiles of CD166, CD29, CD105, CD73, CD44 and CD90 were evaluated with respect to isotype control (gate R2).
The following antibodies were used: PE anti-human CD166 antibody (Biolegend, 343,903), PE anti-human CD29 antibody (Biolegend, 303,003), PE anti-human CD105 antibody (Biolegend, 323,205) PE anti-human CD73 (Ecto-5′-nucleotidase) antibody (Biolegend, 344,003), PE anti-human CD44 antibody (Biolegend, 338,807), PE anti-human CD90 (Thy1) antibody (Biolegend, 328,109), PE mouse IgG1, κ isotype control antibody (Biolegend, 400,111), and human BD Fc block, (BD, 564,220).
Cytokine/chemokine bead assay in vitro
For condition medium (CM) analysis, cells were washed three times with PBS (−), cultured in serum-free α-MEM medium for two days, and used for analysis. Cytokine/chemokine analysis of CM (n = 6) was performed using antibody immobilization on magnetic beads. The CM sample was centrifuged at 13,000g and 4 °C for 5 min, and the resulting supernatant was used for measurement. Concentrations of 40 target proteins in CM were measured using a Milliplex® MAP Cytokine/Chemokine Magnetic Bead Panel kit and Luminex® technology (Merck Millipore). Procedures were performed according to the manual (HCYTOMAG-60K.pdf). Pretreated sample was added at 25 μl per well, and measurements were taken three times. Standard solution was prepared as stated in the manual, seven concentrations were prepared in a fivefold serial dilution series, and three measurements (replicates per analysis) were performed. The 41 target proteins were EGF, FGF-2, Eotaxin, TGF-α, G-CSF, Flt-3L, fractalkine, IFNα2, IFNγ, IL-10, GRO,MCP-3, IL-12P40, MDC, IL-12P70, PDGF-AA, IL-13, PDGF-AB / BB, IL-15, sCD40L, IL-17A, IL-1RA, IL-1α, IL-9, IL-1β, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, RANTES, TNFα, TNFβ, and VEGF (Fig. 3).
CMAs cell aging study in vitro
Cellular senescence studies on mature adipocytes were conducted to confirm the safety of the CMAs prepared for patients. A characteristic of tumorigenic cells is proliferation without stopping. This test is therefore conducted when the cells are cultured to confirm that cells stop growing. CMAs were cultured until growth arrest was noted for six of the 25 patients. The population doubling level (PDL) was also measured (Fig. 4).
Clinical application of the CMAs + condition medium and fat graft (CMAM-FGs) in-vivo
A breast-shaped tissue expander was inserted under the pectoralis major muscle and subcutaneously on the affected breast, marked at the location of the healthy breast, and the regular saline solution was injected through the port of the tissue expander, gradually expanding the breast to a size similar to the healthy breast over 6 months. The volume of the healthy breast was defined preoperatively as the breast volume to be achieved during the reconstruction. In the first transplant procedure, half of the saline solution was drawn out from the tissue expander and CMAM-FG was injected around the outside of the capsule of the expander using a cannula with a diameter of 1.2–2.5 mm. In the second procedure, after removal of the expander, the capsule (fibrous tissue) was also removed. The removal of the capsule allows for more blood circulation and improves CMAM-FG engraftment. CMAM-FG was then injected subcutaneously and into the internal cavity created after removal of the expander. The ratio of CMA to autologous fat was determined based on the results of transplantation experiments on nude mice and subsequent partial breast defects and augmentation procedures, which showed ideal engraftment rates at a volume ratio of 1:100. The amount of CMAs for unilateral transplants ranged from 25.74 × 107 to 38.61 × 107 (33.07 × 107 ± 4.29 × 107), and the amount of CMAs for bilateral transplants ranged from 60.06 × 107 to 64.35 × 107 (62.21 × 107 ± 3.03 × 107). Total unilateral injection volumes were 355 to 870 cc (564.52 ± 118.92). Total bilateral injection volumes were 870 to 1336 cc (avg 1103.00 ± 329.51). CM accounted for 20% of the total fat content. Patients with unilateral cases underwent two transplants, and those with bilateral cases underwent three transplants (Table1; Fig. S1). The patient’s BMI was measured pre and post operation and no significant changes were noted. Pre-Op BMI was 22.8 ± 2.5 and Post-Op BMI was 22.4 ± 2.2. No significant difference (P = 0.12) was found. It is also to be noted that the average obtained was similar to the average BMI of Japanese women (40–69 years old) of 22.6 as reported in 2016 the National Health and Nutrition Survey by the Ministry of Health, Labor and Welfare of Japan48.
Immunofluorescence or immunohistochemical analysis of regenerated fat after CMAM-FGs engraftment in-vivo
Biopsies of reconstructed fat six months to one year after transplantation were analyzed histologically. Tissue samples were fixed at 4 °C in 4% PFA, processed, and embedded in paraffin. Transverse sections (4 μm) were placed on MAS-coated slides (Matsunami Glass Ind. Ltd.) for either immunofluorescence or immunohistochemistry.
1. Expression of perilipin as lipid droplet-associated protein.
Anti-perilipin (guinea pig) (Fitzgerald Industries International, 20R-PP004, MA, USA) (4 °C/1 h, 1:200) was used as the perilipin primary antibody, and Alexa Fluor 488 goat anti-guinea pig IgG (InvitrogenA11073, CA, USA) (room temperature/1 h) was used as the secondary antibody. Encapsulation was achieved using Invitrogen™, SlowFade™, Gold Antifade Mountant with DAPI (Sigma-Aldrich) for nuclear staining. Images were examined using a Zeiss LSM510 laser scanning microscope (Fig. 5A).
2. Detection and characterization of macrophages (CD68), angiogenesis (cell surface marker of differentiation: CD31, CD34), and cell proliferation marker (Ki67)46.
Primary antibodies were incubated overnight at 4 °C. The primary antibodies used were rabbit monoclonal anti-human CD31 (1:200, Abcam: ab76533), mouse monoclonal CD34 anti-human (1:200, Cell Signal, TEC 3569S), mouse monoclonal CD68 anti-human macrophage (1:500, Abcam, ab955), and rabbit monoclonal anti-human Ki 67(30-9) (Roche, 518102456). After treatment with biotin-labeled secondary antibody (LSAB2, DAKO K0609) at room temperature for 30 min, the sections were stained using DAB (Fig. 5B–E).
MRI images were used to confirm the engraftment of cultured fat. T1 and T2-weighed images were taken in the axial and sagittal sections. Diffusion-weighted images were also taken for the axial section. T2-Fat-Saturation-weighted images were taken in the coronal section.
All patients underwent MRI examinations annually after transplantation, and the images were evaluated by one breast surgeon and two radiologists at other facilities. These evaluations indicated full-layer fat formation for all the patients.
Data are shown as the mean ± SEM from at least six independent experiments. For cytokine/chemokine analysis, the concentration of the target protein (X) was calculated from the calibration curve using Master Plex® QT analytical software (Hitachi Solutions) and the five-parameter logistic function. Functional motion field imaging was used to show median fluorescence intensity.
Statistical analysis using a one-way ANOVA with Dunnett’s test was performed to compare the differences between MCP-1 and other cytokines or chemokines after checking for normality of these data with a Shapiro–Wilk test. Statistical significance was considered as p < 0.01 (two-tailed), and all values are expressed as mean ±SD. Statistical software SPSS Statistics 26 (IBM Corporation, Armonk, NY, USA) was used for these analyses.
Patient satisfaction scales
The VAS of patient satisfaction is a horizontal line 10 cm long. At the beginning and end are two descriptors representing the minimum and maximum limits of satisfaction (i.e., no satisfaction and total satisfaction)47. The patients mark their level of satisfaction on the 10 cm line. For reference there are also five levels of human facial expressions on top of the line.
In the satisfaction survey conducted in September 2023 among patients who had undergone surgery 3–6 years earlier. The scale used was 0–100, 100 expressing the highest level of satisfaction.
All patients were explained the purpose and scope of this study and they all provided written informed consent to participate in it.