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A novel cryopreservation solution for adipose tissue based on metformin

Stem Cell Research & Therapy volume 16, Article number: 20 (2025) Cite this article

Abstract

Background

Autologous fat grafting (AFG) often needs multiple sessions due to low volume retention. Young adipose tissue demonstrates a more pronounced therapeutic effect; thus, the cryopreservation of adipose tissue of young origin is particularly crucial. This study investigated the protective effect of a new cryopreservation solution combining trehalose, glycerol, and metformin on adipose tissue.

Methods

This study initially examined the effect of various concentrations of metformin (0, 1, 2, 4, and 8 mM) on oxidative damage in adipose tissue to identify the optimal concentration. Subsequently, 1.5 mL of fresh human adipose tissue was subjected to freezing using trehalose + glycerol (TG group), trehalose + glycerol + metformin (TGM group), and the common cryoprotectant dimethyl sulfoxide (DMSO) + fetal bovine serum (FBS) (DF group). Samples were cryopreserved in liquid nitrogen for 2 weeks. After thawing, 1 mL of adipose tissue from each group was transplanted subcutaneously into the backs of nude mice. The cryoprotective effects on adipose tissue viability were evaluated during transplantation one month after transplantation.

Results

The 2 mM concentration of metformin exhibited the lowest reactive oxygen species (ROS) level (29.20 ± 1.73) compared to other concentrations (P < 0.05). Cell proliferation and migration assays also supported the superior performance of the 2 mM concentration. Apoptotic analyses of stromal vascular fraction (SVF) cells showed the lowest levels in the 2 mM group. Compared to other cryopreservation groups, the adipose tissue in the TGM group closely resembled fresh adipose tissue in terms of gross structure and histological characteristics, with the lowest apoptosis rate of SVF cells. In vivo analysis revealed the highest tissue retention rate in the TGM group, with histological examination indicating robust structural integrity.

Conclusion

The TGM cryopreservation solution, containing metformin, greatly preserves adipose tissue, reduces apoptosis, and improves tissue retention rates. This solution was non-toxic and safe, making it well-suited for tissue cryopreservation in clinical settings.

Background

Autologous fat grafting has become a widely utilized procedure in plastic surgery due to its diverse sources and high biocompatibility. It is typically employed for facial rejuvenation and body contouring [1]. However, despite its benefits, the absorption rate of transplanted adipose tissue remains relatively high, ranging from 30 to 70%. Thus, often larger quantities of adipose tissue are needed to achieve optimal therapeutic outcomes [2]. Moreover, repeated liposuction procedures increase the economic burden and safety risks [3]. Previous studies have indicated that younger patients generally have better-functioning and more vibrant adipose tissue, as well as greater subcutaneous fat [1, 4, 5]. Preserving autologous adipose tissue offers a reliable approach, enabling the possibility of “one-time liposuction, multiple transplantations”. Furthermore, preserved adipose tissue serves as a dependable source for adipose-derived stem cells (ADSCs) in the realms of tissue engineering and regeneration [3, 6].

According to Mazur’s dual-factor theory [7], cryoprotectants (CPAs) are indispensable in cell/tissue cryopreservation as they inhibit ice crystal formation and mitigate the damage caused by freeze-thaw, including osmotic damage [8,9,10]. Moreover, freezing elevates cellular ROS levels, causing oxidative stress, cellular DNA damage, protein damage, and lipid peroxidation [11, 12], finally leading to cell apoptosis. Adipose tissue is a complex tissue primarily composed of mature adipocytes and stromal vascular components, necessitating challenging and complex cryopreservation compared to simple cells and tissues [1]. Currently, there is no standardized cryoprotectant for preserving and storing human adipose tissue. Conventional cryoprotectants often contain DMSO, which is toxic to some extent, and the process of DMSO removal is intricate [13, 14]. Therefore, it is needed to explore safe and non-toxic cryoprotectants with clear compositions.

Trehalose is a non-toxic and biodegradable macromolecule that has demonstrated effectiveness in the cryopreserving of various tissues and cells [15]. However, its application is constrained by the osmotic effects associated with trehalose [16, 17]. Glycerol, a permeable CPA that is safe for humans at low concentrations, acts synergistically with trehalose [18, 19]. While the combination of trehalose and glycerol has proven to be effective in cryopreservation, it does not address oxidative stress [20]. Previous studies have indicated that adding antioxidants to trehalose or glycerol can decrease the cellular levels of ROS [21]. Metformin, a medication commonly used for type 2 diabetes, not only controls blood glucose but also exhibits antioxidant and anti-aging properties and inhibits lipolysis [22,23,24]. Previous studies have shown that metformin can enhance antioxidant enzyme activity and the expression of type II enzymes by promoting the activation of adenosine 5’-monophosphate (AMP)-activated protein kinase (AMPK) and the transcriptional activity of brain and muscle Arnt-like protein-1 (Bmal1)/nuclear factor E2-related factor 2 (Nrf2) [25]. Furthermore, studies have demonstrated that metformin reduces oxidative stress in ADSCs and improves mitochondrial dysfunction through the AMPK pathway, thereby alleviating adipose tissue dysfunction [26]. Additionally, metformin has been shown to mitigate oxidative stress during freezing by activating the AMPK pathway and increasing the expression of relevant antioxidant enzymes [27, 28]. Utilizing a mouse model of lymphedema, our team previously demonstrated that metformin can effectively reduce inflammation and fibrosis by activating the AMPK signaling pathway [29]. These characteristics suggest the potential use of metformin for adipocyte cryopreservation. Thus, metformin not only treats diabetes, but also emerges as a promising candidate for addressing other medical conditions and facilitating tissue cryopreservation. Currently, there is no report on the use of metformin as a CPA for adipose tissue.

Therefore, we propose a novel cryoprotectant combination comprising trehalose, glycerol, and metformin (TGM) for adipose cell cryopreservation. This endeavor seeks to provide a safe and more effective CPA formulation for adipose cryopreservation in the clinical setting.

Materials and methods

Harvest and preparation of adipose tissue

Human adipose tissue were obtained from healthy adult females (aged 23–40 years, BMI median = 25.15 kg/m2) who underwent abdominal or thigh liposuction at the Department of Burn Plastic Surgery of Zunyi Medical University (Table S1). Written informed consent was obtained from all patients. This study was approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University and complied with the principles of the Declaration of Helsinki.

Clinically obtained liposuction aspirates were transported to the laboratory within 1 h under cryogenic conditions, and the swollen fluid was preliminarily cleared using quiescent methods. As shown in Fig. 1, the fat aspirate was mixed with phosphate buffer in a 1:1 volume ratio and centrifuged at 1200 g for 3 min. The lower solution and upper oil droplets were removed. This process was repeated 3–5 times, and the purified adipose tissue in the middle layer was retained for subsequent experiments.

Fig. 1
figure 1

Schematic diagram of the cryopreservation process and experimental procedures. Fat obtained through aspiration was centrifuged at 1200 g for 3 min to isolate the middle adipose tissue layer. This tissue was then mixed with selected cryoprotectants in a 1:1 volume ratio and stored in liquid nitrogen. After 2 weeks, the tissue was thawed and eluted for subsequent in vivo and in vitro studies

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Cryopreservation of adipose tissue

For CPA preparation, we used the following groups: (1) blank: phosphate buffer saline (PBS) (Solarbio, Beijing, China); (2–6) 0, 1, 2, 4, and 8 mM metformin (Medchem Express, China) + 1 M trehalose (Sigma, Germany) + 20% glycerol (Solarbio, Beijing, China) (Met + Tre + Gly); (7) 90% fetal bovine serum (Gibco, USA) and 10% dimethyl sulfoxide (Sigma, Germany) (FBS + DMSO). Adipose tissue samples were aliquoted into 5 mL sterile cryogenic vials, with each tube containing 1.5 mL of samples. An equal volume of cryoprotective solution was added to each tube, mixed uniformly, and left at room temperature for 10 min.

The cryovials were frozen in a gradient cooling cassette filled with isopropanol, and kept at 4 °C for 30 min, transferred to a -20 °C refrigerator for 4 h, and then transferred to a -80 °C refrigerator for 24 h, and finally stored in liquid nitrogen for 2 weeks.

Thawing and elution

Samples were taken out of liquid nitrogen and immediately placed in a 37 °C water bath for 5 min for rapid thawing. An equal volume of PBS was slowly added to the cryopreserved tissue for washing. The mixed samples were centrifuged at 1000 rpm for 3 min to separate and remove the liquid layer, retaining the middle layer. This process was repeated 2–3 times to thoroughly remove the CPAs.

Isolation of SVF cells and ADSCs

The SVF was obtained from rewarmed adipose tissue. Briefly, the adipose tissue was enzymatically digested with 0.075% collagenase I at 37 °C for 50–60 min, followed by centrifugation at 1200 g for 5 min to remove the upper oil droplets. The remaining liquid was then sequentially filtered through 100-µm and 400-µm mesh filters before undergoing a second round of centrifugation at 1200 g for 5 min. The resulting pellet containing the cells was resuspended in a DMEM culture medium (Gibco, USA) after discarding the supernatant.

ADSCs were obtained after collagenase digestion of fresh adipose tissue that had not been cryopreserved. The extraction procedure was the same as that of SVF cells as previously described. Suspensions of ADSCs were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin (Solarbio, Beijing, China) and incubated at 37 °C in a 5% CO2 incubator, and subcultured for three passages (P3).

Measuring intracellular ROS levels of ADSCs

P3 ADSCs were seeded into 96-well plates at a concentration of 5 × 104 cells/mL and sorted into 5 groups based on metformin concentrations (0, 1, 2, 4, and 8 mM). Following a 24-hour incubation period, the cells were exposed to DMEM containing 10 µmol/L fluorescent probe DCFH-DA at 37 °C for 20 min. Subsequently, cells were rinsed with serum-free DMEM and subjected to hypoxic conditions (5% O2) for 2 h. The mean fluorescence intensity (MFI) of each well was quantified using a multimode microplate reader (Bio-Tek, USA).

Mitochondrial activity detection in ADSCs

P3 ADSCs were seeded at a density of 5 × 104 cells/mL in 6-well plates and divided into two groups: PBS and 2 mM metformin. After 24 h of incubation, cells were exposed to DMEM containing 100 nM MitoTracker Red CMXRos (Beyotime, C1035) at 37 °C for 30 min. Subsequently, cells were washed with serum-free DMEM and fixed with a fixation solution containing 4% paraformaldehyde (Solarbio, China). Finally, the mounting medium containing DAPI (Solarbio, China) was applied for fluorescence attenuation sealing. Imaging was conducted using Cell Imaging Multimode Readers (Bio-Tek Cytation 5, USA).

Senescence detection of ADSCs

P3 ADSCs were seeded in 6-well plates at a density of 5 × 104 cells/mL. The cells were divided into three groups: PBS control, 2 mM metformin, and 200 µM H₂O₂. After incubating for 24 h, cells were fixed at room temperature for 15 min using a β-galactosidase staining fixative (Beyotime, C0602). Subsequently, cells were washed three times with PBS, and a staining working solution (Beyotime, C0602) was added. Cells were then incubated overnight at 37 °C. Finally, the results of staining were observed and recorded using an inverted phase-contrast microscope (Olympus, Japan).

RNA extraction and real-time quantitative polymerase chain reaction (RT-PCR)

P3 ADSCs were seeded in 6-well plates at a density of 5 × 104 cells/mL and divided into three groups: PBS control, 2 mM metformin, and 200 µM H₂O₂. Total RNA from each group was extracted using TRIzol™ reagent (Invitrogen, USA) following the manufacturer’s instructions. Following extraction, complementary DNA (cDNA) was synthesized and amplified using the QuantStudio™ Design & Analysis Software system (Thermo Fisher Scientific, USA). Detailed information regarding the primers is provided in Table S2.

ADSC proliferation

A cell counting kit-8 (CCK-8) assay (Tongren, Japan) was employed to evaluate cell proliferation. Initially, P3 generation ADSCs were seeded into 96-well plates (5000 cells/well), and divided into five groups of 0, 1, 2, 4, and 8 mM metformin. Cells were incubated for 24, 48, and 72 h. Subsequently, 10 µL of CCK-8 solution was introduced to the culture medium, and cells were incubated at 37 °C for 2 h. Colorimetric assessment was conducted at 450 nm utilizing a full-wavelength microplate reader (Bio-Tek, USA) to acquire the optical density (OD) value.

Apoptosis assay

The SVF cell suspension was centrifuged at 1000 r/min for 5 min. The pellet was washed and centrifuged again with 1 mL of PBS. Then, 1 × binding buffer was added to remix, and the cell concentration was adjusted to 5 × 106 cells/mL. 100 µL of the prepared mixture was taken, transferred to a flow cytometry tube, 5µL of Annexin V/FITC (Solarbio, Beijing, China) was added, mixed well, and dark incubated at room temperature for 5 min. Then, 5 µL of propidium iodide solution and 400 µL of PBS were added, and immediately a flow cytometer was used for detection (Beckman, USA).

Fat transplantation model

All animal experiments were conducted following the Ethical Principles of Animal Welfare. The work has been reported following the ARRIVE guidelines 2.0. Twenty-four male BALB/c nude mice (5 weeks old, weighing 16–22 g) were purchased from Zhejiang Weitong Lihua Laboratory Animal Technology Co., Ltd (SCXK (Zhe) 2019-0001) and randomly assigned to 4 groups (6 mice/12 sides per group): PBS, 1 M trehalose + 20% glycerol (TG), 1 M trehalose + 20% glycerol + 2 mM metformin (TGM), and 90% FBS + 10% DMSO (DF). Anesthesia induction was achieved using 4–5% isoflurane, followed by maintenance at 1–3% isoflurane concentration. Following anesthesia, 0.5 ml of prepared fat tissue was injected into each side of the nude mouse dorsum. After 1 month, the mice were euthanized by cervical dislocation, and the grafted fat samples were collected and weighed, and their volumes were measured using the liquid overflow method. The weight of the samples was measured using an electronic balance. Graft volume retention was calculated as the harvested graft volume divided by 0.5, multiplied by 100. Graft weight retention was calculated as the harvested graft weight divided by the initial weight, multiplied by 100.

Histological analysis

The grafted fat samples were fixed in a 10% paraformaldehyde solution, embedded in paraffin, and sectioned for histological examination. Histological analysis was conducted using hematoxylin and eosin (H&E) staining. After deparaffinization in xylene and dehydration through a series of ethanol gradients, tissue sections were stained with hematoxylin for 5 min, followed by brief differentiation in hydrochloric acid alcohol and subsequent bluing in running tap water. Subsequently, sections were counterstained with eosin for 5 min, dehydrated in graded ethanol, cleared in xylene, and mounted in neutral resin. Slide processing and observation were conducted using Slideview VS200 ASW (Olympus, Japan).

Immunohistochemistry

The paraffin sections of the fat grafts were deparaffinized at 62 °C for 4 h. Following deparaffinization and antigen retrieval, a catalase blocker was added dropwise. After a 10-minute incubation, the sections were rinsed with PBS at room temperature. Subsequently, the sections were incubated with a non-specific staining blocker. For immunofluorescence analysis, the sections were incubated overnight at 4 °C with the primary antibody against perilipin (GeneTex, USA). Then, the sections were dark incubated with the secondary antibody(Abcam, UK) at room temperature for 45 min. Subsequently, they were washed three times with pure water. Finally, DAPI staining solution was added dropwise, dark incubated for 2 min, and washed 3 times. The anti-fluorescence quencher was added to mount, and the tablets were observed and recorded. Next, for immunohistochemical analysis, the sections were incubated with the primary antibody Anti-CD31 (Ab182981, Abcam, UK ) at 4 °C overnight, followed by PBS washing. Subsequently, the sections were incubated with the secondary antibody (Abcam, UK ) at room temperature for 30 min. After washing, DAB chromogenic solution was added dropwise, and staining lasted for 5 min. Thereafter, counterstaining with hematoxylin was done after rinsing with PBS. Finally, the sections underwent dehydration using a concentration gradient of ethanol. Afterwards, the samples were treated with xylene and mounted with neutral resin. All images were processed using Slideview VS200 ASW software (Olympus, Japan).

Statistical analysis

The experimental data were analyzed using SPSS version 29.0. Following the normality test, one-way analysis of variance (ANOVA) was used to compare groups. Data are presented as mean ± standard deviation, with significance set at P<0.05.

Results

The optimal metformin concentration for cryopreservation was 2 mM

The effect of various concentrations of metformin on ROS in ADSCs was investigated. The results revealed that intracellular ROS levels decreased in all metformin-containing groups compared to the control group (0 mM metformin), indicating the potent antioxidant capacity of metformin. Among the groups, the 2 mM concentration group exhibited the strongest ability to reduce ROS levels (Fig. 2A). Concurrently, we examined the effect of 2 mM metformin on mitochondrial activity and cellular senescence in ADSCs. The results showed that this concentration of metformin did not significantly affect mitochondrial activity in ADSCs (Fig. S1). In β-galactosidase staining assays, the 2 mM group, similar to the PBS group, did not exhibit a significant number of senescent cells (Fig. S2A, B). Additionally, the expression levels of P53 and P21 were lower in cells from the PBS and 2 mM groups compared to the senescence group (H2O2 group) (Fig. S2C). Subsequently, we explored whether metformin, as a CPA, exerted cytotoxic effects. Cell proliferation assays demonstrated that after 24 h, the proliferation rate of ADSCs in the low-concentration group (1 and 2 mM metformin) was notably higher than that in other groups. However, as the concentration increased, cell proliferation exhibited a trend toward inhibition (Fig. 2B, C). Notably, high concentrations (8 mM group) significantly inhibited the migration of ADSCs (P < 0.05) (Fig. 2D, E). These findings suggest that low concentrations of metformin do not induce significant cell toxicity and can enhance cell proliferation. Conversely, high concentrations of metformin adversely affected cell migration.

Fig. 2
figure 2

Screening for optimal concentrations of metformin. (A) Intracellular ROS levels in ADSCs across different concentrations of metformin showed a significant reduction at 2 mM compared to other groups. (B) Proliferation trends of ADSCs observed over 72 h. (C) Quantitative analysis of changes in adipose stem cell proliferation from 0 to 72 h. (D) Migration trends of ADSCs from 0 to 24 h; scale bar = 500 μm. (E) Quantitative analysis of migration changes of ADSCs from 0 to 24 h. (F) Apoptosis rates of adipose tissue SVF cells measured via flow cytometry. (G) Quantitative analysis of the apoptosis rate of SVF cells, * indicates a significant difference compared to the control group with P < 0.05; # indicates a significant difference between groups with P < 0.05

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To identify the optimal concentration of metformin for cryopreservation, the apoptosis rates of SVF cells in adipose tissues were assessed after exposure to different concentrations of metformin. The results indicated lower apoptosis rates in the low-concentration group, with the lowest rate observed in the 2 mM concentration group. Higher concentrations increased the apoptosis rate (Fig. 2F, G).

Based on these outcomes, we selected 2 mM as the ideal concentration of metformin for cryopreservation. This concentration, combined with 1 M trehalose and 20% glycerol, formed a novel CPA combination for subsequent experiments.

Improved morphology and reduced apoptosis rate in the TGM group

The extracted adipose tissues were divided into 4 groups and cryostored in liquid nitrogen for 2 weeks with different preservation solutions: PBS, TG (1 M trehalose + 20% glycerol), TGM (1 M trehalose + 20% glycerol + 2 mM metformin), and DF (10% DMSO + 90% FBS). They were then rewarmed and thawed for subsequent experiments (Fig. 1).

Fresh adipose tissue was used as the control to assess its appearance after freezing. The fresh fat displayed a full and compact structure, with bright yellow color, and no visible oil droplets. Compared to other cryopreservation groups, the TGM group exhibited a denser tissue structure and fewer oil droplets, with greater similarity to fresh fat. Conversely, the remaining cryopreservation groups showed looser tissue structure, darker color, and evident oil droplets (Fig. 3A). Electron microscopy revealed that the TGM group had more intact fat cells attached to connective tissue, displaying uniformity akin to fresh adipose tissue. In contrast, other cryopreserved groups exhibited uneven adipocyte sizes, detachment, and cell death, exposing connective tissue (Fig. 3B). Overall, the TGM group demonstrated superior cryopreservation capability. The TGM group (31.60 ± 1.21%) and DF group (33.20 ± 1.08%) had lower apoptosis rates compared to other groups, with no significant difference between them (Fig. 3C, D). In summary, TGM showed enhanced cryopreservation ability, leading to improved adipose tissue morphology and reduced apoptosis rates.

Fig. 3
figure 3

Morphological analysis and apoptosis rates of adipose tissue after cryopreservation. (A) Gross morphology of fresh adipose tissue two weeks after being subjected to different cryopreservation protocols. (B) Electron microscopy scan of fresh adipose tissue and adipose tissue after cryopreservation of different cryopreservation solutions for two weeks, scale bar (top): 1 mm, scale bar (bottom): 500 μm. (C) Flow cytometry analysis of the apoptosis rates of SVF cells from adipose tissue cryopreserved with different solutions. (D) Quantitative analysis of the apoptosis rate of SVF cells; *, indicates a comparison with the PBS group, P < 0.05; #, indicates a comparison between groups, P < 0.05

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Enhanced retention and histological integrity of adipose tissue in the TGM group after transplantation

To assess the retention rate of adipose tissue after cryopreservation, we transplanted cryopreserved thawed adipose tissue into four groups of nude mice and examined the samples after 1 month (Fig. 4A, B). Adipose tissue in the PBS and TG groups displayed larger oil vesicles compared to the TGM and DF groups, with a noticeable reduction in volume in the PBS group (Fig. 4C). After transplantation, adipose tissue weight and volume were higher in groups supplemented with cryopreservation agent compared to the control group (PBS group). The TGM group exhibited the highest retention rates in terms of body weight (64.80 ± 1.62%) and volume (73.45 ± 4.84%). The TG group was similar to the DF group (Fig. 4D, E). HE staining showed that only the TGM group had uniformly distributed adipocytes without oil vesicle vacuoles, while other groups displayed such vacuoles due to adipocyte necrosis and fusion (Fig. 5A). Evaluation of adipocyte activity using perilipin labeling indicated that the TGM group had the highest level of mature adipocyte activity and structural preservation [30,31,32]. Fewer perilipin protein-positive adipocytes were observed in other groups (Fig. 5B). Overall, the TGM group exhibited superior retention and histological characteristics of adipose tissue following transplantation.

Fig. 4
figure 4

Effect of cryopreserved adipose tissue transplantation. (A) Schematic diagram of cryopreserved adipose tissue injection on both sides of the back of nude mice (n = 6). (B) Schematic diagram for the dissection of samples one month after transplantation. (C) Gross morphology of fat grafts one month after transplantation. (D) Volume retention rate of fat grafts; (E) weight retention rate of fat grafts. *, compared with PBS group, P < 0.05; #, indicates a comparison between groups, P < 0.05

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Fig. 5
figure 5

Morphological assessment of grafted adipose tissue. (A) H&E staining of adipose tissue graft one month after transplantation. Scale bar (upper): 400 μm, scale bar (lower): 200 μm. (B) Immunofluorescence staining of fat graft. Perilipin (green) was used to highlight living adipocytes and DAPI (blue) to indicate cell nuclei. Scale bar: 100 μm

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Enhanced angiogenic capacity of adipose tissue preserved with TGM

The retention rate after adipose tissue grafting is affected by angiogenesis. Previous studies have indicated that in the initial stages of fat grafting, adipocytes receive nutrients and oxygen through plasma diffusion in the surrounding tissues, primarily in the outer region of the grafted fat [33]. Insufficient blood supply to deep adipocytes after transplantation may result in fat necrosis or resorption, underscoring the importance of early revascularization for adipose tissue survival [34]. In this study, we assessed the vascularization capacity of cryopreserved adipose tissue after transplantation. Our findings revealed a significant increase in intra-graft vascularization in the TGM group, with an average of 19.80 ± 1.92 vessels, indicating a higher level of angiogenesis compared to other groups (Fig. 6A, B). This outcome emphasizes the enhanced angiogenesis of adipose tissue after cryopreservation with the TGM solution, suggesting its potential to enhance angiogenesis after transplantation.

Fig. 6
figure 6

CD31 immunohistochemical staining of adipose grafts. (A) CD31 staining of grafts in each group one month after grafting. Scale bar: 200 μm; (B) Mean number of vessels. *, for comparison with PBS group, P < 0.05; #, for comparison between groups, P < 0.05

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Discussion

The development of adipose tissue cryopreservation technology has significantly advanced regenerative medicine and aesthetic transplantation, optimizing the use of adipose tissue to address clinical challenges [35, 36]. This study investigated a novel cryoprotectant combination consisting of trehalose, glycerol, and metformin. The combination was non-toxic and effectively enhanced tissue preservation rates. Trehalose, known for its extracellular cryoprotective capabilities, synergistically improved the cryopreservation process when combined with glycerol [15, 20]. Moreover, metformin, recognized for its antioxidant properties and previously utilized in germ cell cryopreservation [27, 28, 37, 38], significantly improved the effectiveness of this combination at an optimal concentration of 2 mM. Notably, in vivo experiments demonstrated that metformin not only improved the outcomes of adipose tissue cryopreservation but also stimulated angiogenesis in transplanted tissues. These findings support the effectiveness and safety of this cryoprotectant combination.

Trehalose, a non-reducing disaccharide has garnered significant attention for its cryoprotective properties across various organisms and tissues [39,40,41,42]. The protective mechanism of trehalose lies in its extracellular action. It promotes cellular dehydration to inhibit intracellular ice formation and protect cells from damage caused by extracellular ice crystals [15]. Pu et al. explored the cryopreservation effects of trehalose alone on adipose tissue [39]. They demonstrated that at a concentration of 0.25 M, trehalose significantly enhanced cell viability to 1.78 ± 0.33 × 106/mL compared to the absence of CPA. The structure of the cryopreserved adipose tissue remained intact, highlighting the effectiveness of trehalose in preserving tissue integrity. Cui et al. demonstrated that the recovery rate of adipocytes in adipose tissue cryopreserved with 0.5 M DMSO + 0.2 M trehalose (87.1%) was higher than that (80.2%) observed in the 0.25 M trehalose alone group [40]. However, alginate cannot penetrate mammalian cell membranes and enter the intracellular space, greatly affecting its cryopreservation effectiveness [43,44,45]. Although various solutions have been proposed to enhance the permeability of trehalose, their clinical translation remains a complex and formidable challenge. Consequently, the use of trehalose in conjunction with a permeable cryoprotectant may synergistically improve cryopreservation outcomes.

Glycerol is a permeable cryoprotectant that can effectively inhibit the growth of ice crystals, and reduce structural and functional damage to cells caused by ice crystals [46]. Adipose tissue is rich in triglyceride, which has a structure similar to glycerol; thus, adipose tissue may particularly benefit from the cryopreservative properties of glycerol. Studies indicated that glycerol can serve as a structural backbone for adipocytes, facilitating its transport through glycerol transporters [47]. This action helps retain glycerol within adipocytes, preserving the structure and biological function of adipocytes. Furthermore, the non-toxic and non-allergenic nature of glycerol makes it suitable for clinical application. Nevertheless, Zhang et al. found that the viability of cells cryopreserved with glycerol alone was lower than that of cells cryopreserved with a combination of cryoprotective agents [20]. This finding underscored the superior efficacy of combining cryoprotective agents to leverage synergistic effects. Our experiments supported this concept, confirming that the use of a combination of 1.0 M trehalose and 20% glycerol (TG) for cryopreserving adipose tissue provides favorable results. The TG group exhibited better overall tissue structure and vitality compared to the control group. The results of electron microscopy and cell viability of the TG group were similar to those of the DF group.

Oxidative damage during cryopreservation has garnered increasing attention. The freezing process induces cellular oxidative stress, generating ROS that can cause irreversible cellular damage or apoptosis [48, 49]. The integration of antioxidants into cryoprotective solutions remains less studied, particularly for adipose tissue. We introduced metformin, an antioxidant agent with anti-diabetic properties, to the cryopreservation solution. Metformin enhanced metabolic flexibility and cellular glucose uptake, and promoted superoxide dismutase (SOD) production, thereby inhibiting cellular oxidative damage [27]. Bertoldo et al. demonstrated that low doses of metformin do not lower sperm quality in fresh semen, and may even improve the quality of cryopreserved semen [38]. Zhang et al. and Bashghareh et al. demonstrated the protective effects of metformin against oxidative stress on sheep semen and mouse spermatogonial stem cells, respectively [27, 28]. In adipose tissue, ROS can be generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, and mitochondrial oxidative phosphorylation system. SOD and antioxidant enzymes can mitigate ROS burden and serve as antioxidant defenses in adipose tissue [50]. Metformin can alleviate ROS generation during the cryopreservation process by increasing SOD levels, thereby mitigating cell damage. Studies have shown that Nrf2, a member of the basic leucine zipper (bZIP) transcription factor Cap-n-collar subfamily, can modulate the expression of antioxidant proteins to prevent oxidative stress [50]. Using in vitro experiments, Chhunchha et al. indicated that metformin can activate the AMPK signaling pathway, thereby promoting the expression of Bmal1 and Nrf2, along with their antioxidant target genes, facilitating cellular antioxidant defense [25]. This may also be one of the mechanisms by which metformin alleviates adipose tissue damage during the cryopreservation process.

In this study, TGM (trehalose, glycerol, and metformin) cryopreservation solution with 2 mM metformin demonstrated the strongest antioxidant capacity and significantly reduced the apoptosis rate of SVF after cryopreservation. Comparative analyses highlighted the superiority of the TGM solution, closely mirroring the characteristics of fresh adipose tissue in gross examination and electron microscopy. In vivo experiments also revealed that the TGM group exhibited a higher count of adipocytes and new blood vessels and reduced fibrous connective tissue, compared to the TG and DF groups. Additionally, our study assessed the potential cytotoxic effects of metformin, finding that low metformin concentrations (2 mM) did not adversely affect cell proliferation. These findings support the non-toxic nature of the TGM cryoprotective solution for clinical applications. Regarding the cryopreservation dosage, we referred to previous literature and adopted a small sample size for cryopreservation with a 1:1 ratio of tissue to cryopreservation medium [13, 47]. Numerous such small samples were prepared for each group. After further safety and efficacy validation of this method in clinical practice, we will adjust the cryopreservation dosage based on clinical needs.

The mechanisms underlying the survival of adipose tissue grafts are not yet fully understood [51]. Using fluorescent tracking in murine models, researchers observed that stromal cells from donor adipose tissue and host-derived endothelial cells form new vasculature in the adipose tissue graft, facilitating its integration with surrounding tissues [52]. Another study employed human-derived stromal vascular fraction (SVF) to assist in human adipose tissue transplantation into nude mice. Fluorescent in situ hybridization showed that adipocytes in the newly formed tissue contained cells from both human tissue origin and host murine hematopoietic stromal cells [53]. Furthermore, a study utilizing matrix tracing techniques explored the role of macrophage-mediated extracellular matrix remodeling in the survival of adipose grafts. The results indicated that the newly formed extracellular matrix in the grafts originates from both donor and host sources [54]. In summary, the survival of adipose grafts after transplantation depends on growth factors released by donor adipose tissue, vascularization mediated by stromal cells, and the involvement of host cells, collectively promoting graft survival and integration. Therefore, in our experiments, we did not differentiate the sources of newly formed vasculature following adipose transplantation into mice. Further studies are needed to elucidate the origins of these newly formed blood vessels.

Based on previous studies, fresh adipose tissue exhibits superior outcomes after transplantation compared to cryopreserved adipose tissue, demonstrating more intact histological structures and greater cell viability [51]. Our study compared the histological effects of different cryopreservation protocols on adipose tissue to evaluate whether the chosen cryopreservation method is superior to alternative approaches. Although this study did not directly compare cryopreserved adipose tissue with fresh adipose tissue, in vivo results indicated that our cryopreservation method outperforms other methods. Future studies should compare our cryopreserved adipose tissue with fresh adipose tissue to validate and refine our findings. Additionally, studies on adipose tissue cryopreservation cover a range of freezing durations from several days to several months [47]. In this study, we only cryopreserved adipose tissue for 2 weeks. Further studies are needed to assess the efficacy of TGM cryoprotective agents over extended durations and achieve long-term cryopreservation of adipose tissue. Though the results are promising, further clinical studies are necessary to confirm the safety and effectiveness of the TGM cryoprotective solution and develop standardized cryopreservation protocols for adipose tissue.

Conclusion

Our study underscores the use of TGM cryoprotective agents for adipose tissue cryopreservation, offering improved tissue preservation and enhanced antioxidative effects. Metformin, as an integral component, contributes to the superior performance of TGM in maintaining tissue integrity and viability. The non-toxic nature of TGM further enhances its suitability for clinical applications, highlighting its potential in transplantation and regenerative medicine.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

AFG:

Autologous fat grafting

DMSO:

Dimethyl sulfoxide

FBS:

Ffetal bovine serum

ROS:

Reactive oxygen species

SVF:

Stromal vascular fraction

ADSCs:

Adipose-derived stem cells

CPAs:

Cryoprotectants

AMPK:

Adenosine 5’-monophosphate-activated protein kinase

Bmal1:

muscle Arnt-like protein-1

Nrf2:

Nuclear factor E2-related factor 2

PBS:

Phosphate buffer saline

MFI:

Mean fluorescence intensity

cDNA:

complementary DNA

CCK-8:

Cell counting kit-8

OD:

Optical density

H&E:

Hematoxylin and eosin

ANOVA:

one-way analysis of variance

SOD:

Superoxide dismutase

NADPH:

Nicotinamide adenine dinucleotide phosphate

bZIP:

basic leucine zipper

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Acknowledgements

The authors would like to express their gratitude to EditSprings (https://www.editsprings.cn ) for the expert linguistic services provided.

Funding

This study was supported by the Key Projects of Science and Technology Plan of Guizhou Province (ZK2021-011).

Author information

Author notes

  1. Yaping Deng and Xin Liu contributed equally to this work.

Authors and Affiliations

  1. Department of Burns and Plastic Surgery, Affiliated Hospital of Zunyi Medical University, Zunyi Guizhou, 563003, China

    Yaping Deng, Xin Liu, Xichao Jian, Yan Zhang, Yinchi Hou, Suyun Hou, Fang Qi, Shune Xiao & Chengliang Deng

  2. Collaborative Innovation Center of Tissue Repair and Regenerative Medicine, Zunyi City, Guizhou Province, 563003, China

    Fang Qi, Shune Xiao & Chengliang Deng

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  1. Yaping Deng
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Contributions

Conceptualization, XL and CLD; Methodology, YPD, XL, YZ, SYH, SEX and FQ; Validation and formal analysis, YPD, XL, XCJ, YCH, SEX and FQ; Writing—original draft preparation: YPD; Writing—review and editing, FQ and CLD; Supervision, SEX, FQ and CLD; Funding acquisition, CLD. All authors have read and agreed to the published version of the manuscript.

Corresponding authors

Correspondence to Fang Qi, Shune Xiao or Chengliang Deng.

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Ethics approval and consent to participate

The animal experiment with project title “Analysis of novel adipose tissue cryopreservation solution using metformin-trehalose-glycerol” (Project ID: ZMU21-2109-001) was approved by the Laboratory Animal Welfare Ethics Committee of Zunyi Medical University on September 16, 2021.The research protocol for the project “Analysis of novel adipose tissue cryopreservation solution using metformin-trehalose-glycerol” was approved by the Ethics Committee of the Affiliated Hospital of Zunyi Medical University on December 31, 2021, following the Declaration of Helsinki principles. Quasi-batch number: KLLY-2021-124. All participants provided written informed consent before the surgical procedure.

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Deng, Y., Liu, X., Jian, X. et al. A novel cryopreservation solution for adipose tissue based on metformin. Stem Cell Res Ther 16, 20 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13287-025-04142-7

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Keywords

Stem Cell Research & Therapy

ISSN: 1757-6512