Short-term cryoprotectant-free cryopreservation at -20°C does not affect the viability and regenerative capacity of nanofat

Abstract

Nanofat is an autologous fat derivative with high regenerative activity, which is usually administered immediately after its generation by mechanical emulsification of adipose tissue. For its potential repeated use over longer time, we herein tested whether cryopreservation of nanofat is feasible. For this purpose, the inguinal fat pads of donor mice were processed to nanofat, which was i) frozen and stored in a freezer at -20°C, ii) shock frozen in liquid nitrogen with subsequent storage at -80°C or iii) gradually frozen and stored at -80°C. After 7 days, the cryopreserved nanofat samples were thawed and immunohistochemically compared with freshly generated nanofat (control). Nanofat frozen and stored at -20°C exhibited the lowest apoptotic rate and highest densities of blood and lymph vessels, which were comparable to those of control. Accordingly, nanofat cryopreserved at -20°C or control nanofat were subsequently fixed with platelet-rich plasma in full-thickness skin defects within dorsal skinfold chambers of recipient mice to assess vascularization, formation of granulation tissue and wound closure by means of stereomicroscopy, intravital fluorescence microscopy, histology and immunohistochemistry over 14 days. These analyses revealed no marked differences between the healing capacity of wounds filled with cryopreserved or control nanofat. Therefore, it can be concluded that cryopreservation of nanofat is simply feasible without affecting its viability and regenerative potential. This may broaden the range of future nanofat applications, which would particularly benefit from repeated administration of this autologous biological product.

Keywords: angiogenesis; cryopreservation; nanofat; platelet-rich plasma; vascularization; wound healing.

FIGURE 1

Cryopreservation and characterization of nanofat. (A) Macroscopic appearance of fresh nanofat (control) as well as nanofat that was frozen and stored at −20°C, shock frozen (SF) in liquid nitrogen or gradually frozen (GF) with subsequent storage at −80°C. (B, C) Immunohistochemical detection of casp-3⁺ apoptotic cells (B) and their quantitative analysis (C) in fresh nanofat (control; white bar; n = 4) as well as nanofat that was frozen and stored at −20°C (light gray bar; n = 4), shock frozen (SF) in liquid nitrogen (dark gray bar; n = 4) or gradually frozen (GF) with subsequent storage at −80°C (black bar; n = 4). Means ± SEM; *p < 0.05 vs control; ^# p < 0.05 vs. −20°C. (D–G) Immunohistochemical detection of CD31⁺ microvessels (D) and LYVE-1⁺ lymph vessels (E) and their quantitative analysis (F, G) in fresh nanofat (control; white bars; n = 4) as well as nanofat that was frozen and stored at −20°C (light gray bars; n = 4), shock frozen (SF) in liquid nitrogen (dark gray bars; n = 4) or gradually frozen (GF) with subsequent storage at −80°C (black bars; n = 4). Means ± SEM. No significant differences between the groups.

FIGURE 2

In vivo microscopy of healing wounds. (A) Stereomicroscopy of wounds filled with fresh (control) or cryopreserved nanofat on days 0, 3, 6, 10 and 14 (initial wound borders = closed lines; wound borders at the indicated time points = broken lines). (B) Wound area (% of day 0) of wounds filled with fresh (white bars; n = 8) or cryopreserved (black bars; n = 8) nanofat on days 0, 3, 6, 10 and 14, as assessed by stereomicroscopy. Means ± SEM. No significant differences between the groups. (C, D) Intravital fluorescence microscopy of wounds filled with fresh [control, (C)] or cryopreserved (D) nanofat on days 0 and 10. Higher magnification of dotted frames is shown in the right panels. (E, F) Perfused ROIs [(E), %)] and functional microvessel density [(F), cm/cm²] of wounds filled with fresh (white bars; n = 8) or cryopreserved (black bars; n = 8) nanofat on days 0, 3, 6, 10 and 14, as assessed by intravital fluorescence microscopy. Means ± SEM. No significant differences between the groups.

FIGURE 3

Tissue and extracellular matrix formation in healing wounds. (A) HE-stained sections (left panels = overview; right panels = higher magnification) of wounds (borders marked by broken lines) filled with fresh (control) or cryopreserved nanofat on day 14. (B–D) Epithelialization [(B), %], granulation tissue formation [(C), %] and cellular density [(D), mm⁻²] of wounds filled with fresh (white bars; n = 8) or cryopreserved (black bars; n = 8) nanofat on day 14, as assessed by histology. Means ± SEM. No significant differences between the groups. (E,F) Immunohistochemical detection of Col I and III in wounds filled with fresh [control, (E)] or cryopreserved (F) nanofat on day 14. (G,H) Total Col I (G) and Col III (H) ratio (wound/skin) of wounds filled with fresh (white bars; n = 8) or cryopreserved (black bars; n = 8) nanofat on day 14, as assessed by immunohistochemistry. Means ± SEM. No significant differences between the groups.

FIGURE 4

Vascularization and lymphatic drainage of healing wounds. (A) Immunohistochemical detection of CD31⁺ microvessels in wounds filled with fresh (control) or cryopreserved nanofat on day 14. (B) Microvessel density (mm⁻²) of wounds filled with fresh (white bars; n = 8) or cryopreserved (black bars; n = 8) nanofat on day 14, as assessed by immunohistochemistry. Means ± SEM. No significant differences between the groups. (C) Immunohistochemical detection of CD31⁺/GFP⁻ (arrows) and CD31⁺/GFP⁺ (arrowheads) microvessels in wounds filled with fresh (control) or cryopreserved nanofat on day 14. (D) CD31⁺/GFP⁺ microvessels (%) in wounds filled with fresh (white bars; n = 8) or cryopreserved (black bars; n = 8) nanofat on day 14, as assessed by immunohistochemistry. Means ± SEM. No significant differences between the groups. (E) Immunohistochemical detection of LYVE-1⁺ lymph vessels in wounds filled with fresh (control) or cryopreserved nanofat on day 14. (F) Lymph vessel density (mm⁻²) of wounds filled with fresh (white bars; n = 8) or cryopreserved (black bars; n = 8) nanofat on day 14, as assessed by immunohistochemistry. Means ± SEM. No significant differences between the groups. (G) Immunohistochemical detection of LYVE-1⁺/GFP⁻ (arrows) and LYVE-1⁺/GFP⁺ (arrowheads) lymph vessels in wounds filled with fresh (control) or cryopreserved nanofat on day 14. (H) LYVE-1⁺/GFP⁺ microvessels (%) in wounds filled with fresh (white bars; n = 8) or cryopreserved (black bars; n = 8) nanofat on day 14, as assessed by immunohistochemistry. Means ± SEM. No significant differences between the groups.