A Novel Porcine Model for Future Studies of Cell-enriched Fat Grafting

Abstract

Background: Cell-enriched fat grafting has shown promising results for improving graft survival, although many questions remain unanswered. A large animal model is crucial for bridging the gap between rodent studies and human trials. We present a step-by-step approach in using the Göttingen minipig as a model for future studies of cell-enriched large volume fat grafting.

Methods: Fat grafting was performed as bolus injections and structural fat grafting. Graft retention was assessed by magnetic resonance imaging after 120 days. The stromal vascular fraction (SVF) was isolated from excised fat and liposuctioned fat from different anatomical sites and analyzed. Porcine adipose-derived stem/stromal cells (ASCs) were cultured in different growth supplements, and population doubling time, maximum cell yield, expression of surface markers, and differentiation potential were investigated.

Results: Structural fat grafting in the breast and subcutaneous bolus grafting in the abdomen revealed average graft retention of 53.55% and 15.28%, respectively, which are similar to human reports. Liposuction yielded fewer SVF cells than fat excision, and abdominal fat had the most SVF cells/g fat with SVF yields similar to humans. Additionally, we demonstrated that porcine ASCs can be readily isolated and expanded in culture in allogeneic porcine platelet lysate and fetal bovine serum and that the use of 10% porcine platelet lysate or 20% fetal bovine serum resulted in population doubling time, maximum cell yield, surface marker profile, and trilineage differentiation that were comparable with humans.

Conclusions: The Göttingen minipig is a feasible and cost-effective, large animal model for future translational studies of cell-enriched fat grafting.

Fig. 1.

MR images and delineation of the ROI (subcutaneously implanted bolus fat graft). A, Immediate postgrafting MR images. D, The same areas on day 120. The inset images (B, C, E and F) show the graft areas magnified with and without delineation for reference. MR, magnetic resonance.

Fig. 2.

MR images with delineation of the ROI (large-volume structural fat graft in the breast area of the pig). This example pig received bilateral structural fat grafting. A, Immediate postgrafting MR images. B, The same areas on day 120. Note the nipple, which serves as a reference point for setting the cranial/caudal borders. MR, magnetic resonance.

Fig. 3.

The mean SVF cells/g fat obtained by syringe liposuction from the back, neck, and abdomen regions were 4.92 × 10⁵ ± 1.96 × 10⁵, 6.08 × 10⁵ ± 0.75 × 10⁵, and 7.72 × 10⁵ ± 2.10 × 10⁵ SVF cells/g fat, respectively, and those obtained by surgical excision were 9.93 × 10⁵ ± 2.32 × 10⁵, 10.8 × 10⁵ ± 2.82 × 10⁵, and 11.4 × 10⁵ ± 3.40 × 10⁵ SVF cells/g fat, respectively (n = 4). In contrast, as shown in Table 1, suction-assisted liposuction from the abdomen in larger volumes yielded a mean of 9.95 × 10⁵ SVF cells/g fat. This difference could be explained by the different harvesting techniques, but most likely because the pigs undergoing syringe liposuction were exsanguinated before the procedure, and thus, a smaller amount of blood cells were mixed with the lipoaspirate, that is, in the SVF.

Fig. 4.

ASC morphology in pPPL cultured cells. Populations of plastic adherent, ASCs occurred from both lipoaspirate and excised adipose tissue with an elongated, spindle shaped appearance. After passage and reseeding with 5,000 cells/cm², confluency was reached after 5–7 days with the use of 10% pPPL. These ASCs also had the ability to become “overconfluent” before reaching replicative senescence. Magnification: 10× original magnification. For full figure of the ASC morphology, see Supplemental Digital Content 3.

Fig. 5.

Differentiation capacity of porcine ASCs. Cells were expanded in culture with 5 different growth supplements before trilineage differentiation was induced. Oil red O, Alcian Blue, and Alizarin red S staining were used to visualize lipid vacuoles, proteoglycans and calcium complexes, respectively. Here only ASCs cultured in pPPL are shown. For the full figure, see Supplemental Digital Content 4. Adipogenic differentiation: 20× original magnification. Chondrogenic and osteogenic differentiation: 10× original magnification.

Fig. 6.

Growth curves of ASCs cultured in different growth supplements (n = 3). The fit lines were calculated with the following formula: y = 10^{ax + b} (where a is the slope, and b is the intercept), with x = (log10[y] – b) / a. Lower right: The mean cell density of all growth supplements over time.

Fig. 7.

Mean cell densities are shown for the ASCs during the plateau phase (n = 3). Significant differences were found among all groups when multiple comparisons were performed (1-way ANOVA). The highest Bonferroni-adjusted P value is shown. 10% FBS = 7.19 × 10⁴ ± 0.97 × 10⁴ ASC/cm², 20% FBS = 17.70 × 10⁴ ± 0.94 × 10⁴ ASC/cm², pPPL = 12.10 × 10⁴ ± 1.29 × 10⁴ ASC/cm², pHPL = 8.72 × 10⁴ ± 1.63 × 10⁴ ASC/cm², and PS = 5.20 × 10⁴ ± 0.80 × 10⁴ ASC/cm².