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Background Long-term preservation of adipose tissue is crucial for clinical applications. Researchers should consider both efficiency and biosafety when choosing a cryoprotective agent (CPA) for adipose tissue preservation. Glycerol has been applied as a nontoxic CPA for multiple tissues but not adipose tissue. We aimed to evaluate the efficacy of glycerol as a CPA for adipose tissue cryopreservation. Methods Fresh human adipose tissues were obtained from patients who underwent liposuction and divided into 1 mL samples. Each sample was randomly mixed with 1 mL of CPA: 60–100% glycerol, 0.25 mol/L trehalose or DMSO + FBS and cryopreserved in − 196 °C liquid nitrogen for one month. After thawing and elution, the tissues were immediately evaluated for activity and structural integrity in vitro. Then, 0.2 mL of each sample was transplanted subdermally to the nude mouse dorsum and harvested after one month for histological examination to assess the effect of the cryopreserved fat in transplantation. Results After cryopreservation, the samples treated with DMSO + FBS, trehalose, 60% and 70% glycerol had a more integrated structure than the samples in other groups. Tissues preserved with 70% glycerol had the highest G3PDH activity of 24.41 ± 0.70, comparable to 24.76 ± 0.48 in fresh tissue (p > 0.05). Adipose-derived stem cells (ASC) viability, proliferation and differentiation capability were also better preserved in 70% glycerol group. In vivo analysis showed that tissue preserved with 70% glycerol had a retention rate of 52.37 ± 7.53%, significantly higher than other groups. Histological observation demonstrated better structural integrity and viability in 70% glycerol group. Compared to the DMSO + FBS and trehalose groups, the glycerol groups showed lower tissue inflammation. Conclusion Glycerol (70%) is efficient in adipose tissue cryopreservation. Glycerol-based CPAs, which are nontoxic and show biosafety, are a promising solution for clinical tissue cryopreservation.

Background: Tissue ischemia usually leads to necrosis and is a threatening condition associated with reconstructive surgery. Promoting the survival of ischemic tissue is critical for improving clinical outcomes. Although various solutions based on stem cells have been reported, there are still limitations to clinical translation. The aim of this study was to develop an effective method to promote the survival of ischemic tissue.Methods: Adipose-derived CD34+ and CD34- cells were obtained by magnetic bead sorting from the stromal vascular faction (SVF). Adipose-derived stem cells (ADSCs) were collected by subculture. The angiogenic capacities of CD34+ cells, CD34- cells and ADSCs were evaluated in vitro by comparing mRNA and protein expression. Random axial flaps in nude mice were used to evaluate the efficacy of these cells in protecting tissue from necrosis. The effect of these cells in preventing inflammation was also evaluated.Results: Our data suggest that CD34+ cells expressed higher levels of angiogenetic factors and lower levels of inflammatory factors than the other cell types. More vessel branches were formed when human umbilical vein endothelial cells (HUVECs) were treated with conditioned medium from CD34+ cells than conditioned medium from the other cell types. Compared to ADSCs, CD34+ cells showed significantly higher efficacy in promoting tissue survival. More CD31+ cells and higher levels of angiogenic factors were observed in tissues from the CD34+ group than in those from the other groups. Lower levels of the proinflammatory factors TNF-α and IL-1b and higher levels of anti-inflammatory factors were found in the CD34+ group than in the other groups.Conclusion: Adipose-derived CD34+ cells showed better efficacy in improving ischemic tissue survival than ADSCs by reducing tissue inflammation and promoting angiogenesis. CD34+ cells can be obtained easily and may be suitable for clinical applications.

Clinical application of mechanical stretching is a reconstructive method for skin repair. Although studies have reported dermal fibroblast heterogeneity, whether stretching affects individual fibroblast subpopulations equally remains unclear. In this study, we show the changes in dermal structure and papillary fibroblast (Fp) in regenerated human skin. Exhausted skin regeneration caused dermal‒epidermal junction flattening, papillary dermis thinning, and an increase in type III collagen-to-type I collagen ratio, with upregulated hallmarks of aging. Well-regenerated skin displayed a notable increase in the Fp population. Consistent changes were observed in the rat expansion model. Moreover, we found that TGFβ1 expression was especially increased in skin showing good regeneration. Activation of the TGFβ1/SMAD2/3 pathway improved exhausted skin regeneration and resulted in increased collagen content and Fp proliferation, whereas pharmacological inhibition of TGFβ1 action impacted well-regenerated skin. Short-term mechanical stretching that promoted skin regeneration enhanced Fp proliferation, extracellular matrix synthesis, and increased TGFβ1 expression, leading to good regeneration. Conversely, long-term stretching induced premature Fp senescence, leading to poor regeneration. This work shows the mechanism of mechanical stretching in well-skin regeneration that enhances Fp proliferation and extracellular matrix synthesis through the TGFβ1/SMAD2/3 pathway and highlights a crucial role of Fps in stretching-induced skin regeneration.

Background: Tissue ischemia usually leads to necrosis and is a threatening condition associated with reconstructive surgery. Promoting the survival of ischemic tissue is critical for improving clinical outcomes. Although various solutions based on stem cells have been reported, there are still limitations to clinical translation. The aim of this study was to develop an effective method to promote the survival of ischemic tissue. Methods: Adipose-derived CD34+ and CD34- cells were obtained by magnetic bead sorting from the stromal vascular faction (SVF). Adipose-derived stem cell (ADSC) were collected by subculture. The angiogenic capacities of CD34+ cells, CD34- cells and ADSC were evaluated in vitro by comparing mRNA and protein expression. Random axial flaps in nude mice were used to evaluate the efficacy of these cells in protecting tissue from necrosis. The effect of these cells in preventing inflammation was also evaluated. Results: Our data suggest that CD34+ cells expressed higher levels of angiogenetic factors and lower levels of inflammatory factors than the other cell types. More vessel branches were formed when human umbilical vein endothelial cells (HUVECs) were treated with conditioned medium from CD34+ cells than conditioned medium from the other cell types. Compared to ADSC, CD34+ cells showed significantly higher efficacy in promoting tissue survival. More CD31+ cells and higher levels of angiogenic factors were observed in tissues from the CD34+ Group than from the other Groups. Lower levels of the proinflammatory factors TNF-α and IL-1b and higher levels of anti-inflammatory factors were found in the CD34+ Group than in the other Groups.Conclusion: Adipose-derived CD34+ cells showed better efficacy in improving ischemic tissue survival than ADSC by reducing tissue inflammation and promoting angiogenesis. CD34+ cells can be obtained easily and may be suitable for clinical applications.

Background Adipose-derived stem cells (ADSCs) promote tissue regeneration and repair. Cryoprotective agents (CPAs) protect cells from cryodamage during cryopreservation. Safe and efficient cryopreservation of ADSCs is critical for cell-based therapy in clinical applications. However, most CPAs are used at toxic concentrations, limiting their clinical application. Objective The aim of this study is to develop a non-toxic xeno-free novel CPA aiming at achieving high-efficiency and low-risk ADSC cryopreservation. Methods We explored different concentrations of trehalose (0.3 M, 0.6 M, 1.0 M, and 1.25 M) and glycerol (10%, 20%, and 30% v/v) for optimization and evaluated and compared the outcomes of ADSCs cryopreservation between a combination of trehalose and glycerol and the commonly used CPA DMSO (10%) + FBS (90%). All samples were slowly frozen and stored in liquid nitrogen for 30 days. The effectiveness was evaluated by the viability, proliferation, migration, and multi-potential differentiation of the ADSCs after thawing. Results Compared with the groups treated with individual reagents, the 1.0 M trehalose (Tre) + 20% glycerol (Gly) group showed significantly higher efficiency in preserving ADSC activities after thawing, with better outcomes in both cell viability and proliferation capacity. Compared with the 10% DMSO + 90% FBS treatment, the ADSCs preserved in 1.0 M Tre + 20% Gly showed similar cell viability, surface markers, and multi-potential differentiation but a significantly higher migration capability. The results indicated that cell function preservation can be improved by 1.0 M Tre + 20% Gly. Conclusions The 1.0 M Tre + 20% Gly treatment preserved ADSCs with a higher migration capability than 10% DMSO + 90% FBS and with viability higher than that with trehalose or glycerol alone but similar to that with 10% DMSO + 90% FBS and fresh cells. Moreover, the new CPA achieves stemness and multi-potential differentiation similar to those in fresh cells. Our results demonstrate that 1.0 M Tre + 20% Gly can more efficiently cryopreserve ADSCs and is a non-toxic CPA that may be suitable for clinical applications.

Background: Adipose-derived stem cells (ADSCs) promote tissue regeneration and repair. Cryoprotective agents (CPA) protect cells from cryodamage in the process of cryopreservation. Safe and efficient cryopreservation of ADSCs is critical in the clinical application of cell-based therapy. However, most CPAs contain toxic concentrations limiting the possibility of their clinical application. Objective: The aim of this study is to develop a non-toxic xeno-free CPA for ADSCs to achieve high-efficiency and low-risk cryopreservation. Methods: We explored the most efficient concentrations in different concentrations of trehalose (0.3M, 0.6M, 1.0M, and 1.25M) and glycerol (10%, 20%, 30% v/v); then evaluated the outcome of the combination of trehalose and glycerol in ADSC cryopreservation, compared to the commonly used CPA, DMSO (10%) + FBS (90%). All samples were slowly freezed and stored in liquid nitrox for 30 days. The effectiveness was evaluated by the cell viability, proliferation, migration and multi-potential differentiation of ADSCs after thawing. Results: Compared to the CPAs with single reagent, 1.0M Tre + 20%Gly group showed significantly higher efficiency in preserving ADSCs activities after thawing, with better outcome in both cell viability and proliferating capacity. Compared to 10%DMSO+90%FBS, ADSCs preserved in 1.0M Tre + 20%Gly group showed similar cell viability, surface markers and multi-potential differentiation but significantly higher migration capability, indicating that better cell function preservation can be achieved by 1.0M Tre + 20%Gly. Conclusions: 1.0M Tre + 20%Gly can preserve ADSCs with high migration capability and cell viability compared to 10%DMSO+90%FBS and maintain similar stemness and multi-potential differentiation as fresh cells. Our results demonstrate that 1.0M Tre + 20%Gly can achieve highly efficient cryopreservation of ADSCs and is suitable for clinical applications.