Cool Fat,Hot Topic: A Systematic Review on Cryopreservation of Adipose Tissue

Abstract

Autologous fat grafting is increasingly used in plastic, reconstructive, and esthetic surgery. Cryopreservation offers a promising solution for the long-term storage of adipose tissue, enabling multiple grafting sessions while minimizing patient discomfort associated with repeated liposuction for fat harvesting. This systematic review aims to analyze the current literature focusing on factors that influence the outcome of cryopreservation, including the use of cryoprotectants, the cooling and warming rate, the storage temperature, and the enrichment of cryopreserved fat grafts. A systematic search of the PubMed/MEDLINE database up to November 2024 was performed, including original preclinical and clinical studies written in English describing the cryopreservation of unprocessed or mechanically processed adipose tissue (macrofat, microfat, or nanofat). Eligible articles needed to describe the applied cryopreservation protocol, at least the storage temperature. Studies on cryopreservation of adipose-derived stem cells (ASCs), stromal vascular fraction, microvascular fragments, and other isolated components of adipose tissue were excluded. Data on cryoprotectants, cooling and warming rates, storage temperature, and eventual supplementation or enrichment of frozen fat were collected. Of the 679 records identified, 59 met the inclusion criteria. Adipose tissue cryopreservation at −80°C with a cryoprotectant, controlled slow cooling, and fast warming represented the most often applied protocol with encouraging outcomes in maintaining tissue survival and histological structure. Several studies indicated that the supplementation of frozen adipose tissue with ASCs improves tissue survival. Taken together, existing studies present diverse, and to some extent, conflicting results regarding cryopreservation protocols and their effects on adipose tissue viability. Hence, the ideal cryopreservation protocol for autologous fat remains to be established. Moreover, tailored protocols may be necessary for the cryopreservation of fat derivatives, such as nanofat.

Impact Statement

This systematic review provides an overview of factors that crucially determine the outcome of cryopreservation of adipose tissue. These include the use of cryoprotectants, the cooling and warming rate, the storage temperature, and the enrichment of cryopreserved fat. Accordingly, this review can help to establish and further improve protocols for the cryopreservation of fat and fat derivatives. If this succeeds, cryopreservation of adipose tissue may evolve as a standard technique in future clinical practice enabling multiple grafting sessions in plastic, reconstructive, and esthetic surgery while minimizing patient discomfort.

Keywords

adipose tissue cryopreservation fat grafting survival cryoprotectant

Introduction

Autologous fat grafting is increasingly widespread in plastic surgery, both for reconstructive purposes as well as for esthetic and regenerative means. Typical areas of application include the treatment of scars, wounds, congenital and genetic deformities, as well as postsurgical organ reconstruction and contour improvement, for instance, of the breast.^1–7 Moreover, autologous fat grafting is applied in the field of autoimmune diseases, inflammatory conditions, and anti-aging (Fig. 1).^8–11 Indeed, it has been demonstrated that adipose tissue represents an ideal autologous filler and is a rich source of growth factors, adipose-derived stem cells (ASCs), and microvascular fragments with a high regenerative capacity.^12–14 Moreover, it can be harvested from various donor areas of the body by means of liposuction and transplanted without severe immunological responses due to its excellent biocompatibility.^12,15

FIG. 1.

Clinical applications of autologous fat grafting. These include the treatment of scars, wounds, deformities, as well as postsurgical organ reconstruction and contour improvement. Furthermore, autologous fat grafting is widely used for regenerative, anti-aging, and esthetic purposes. Finally, it is also applied in the treatment of autoimmune and inflammatory conditions.

However, fat grafting is also associated with certain problems, such as the unpredictable resorption rate, fibrosis, and microcalcification of the grafted tissue.^16–19 Since the first description of fat grafting by Gustav Adolf Neuber,²⁰ several approaches have been proposed to enhance the survival rate and beneficial effects of grafted fat. In this context, it has been demonstrated that adipose tissue can be enzymatically or mechanically processed to fat derivatives, such as the stromal vascular fraction (SVF) or nanofat, which are characterized by a higher concentration of the abovementioned regenerative factors and cellular components.^21–24 Moreover, the enrichment of fat and/or the tissue at the recipient site with biologically active gels, stem cells or microvascular fragments as well as their preconditioning by means of different stimuli prior to transplantation have been shown to improve the outcome of autologous fat grafting regarding the take rate.^25,26

Despite these improvements, some clinical conditions still require multiple surgical fat grafting sessions to achieve or maintain the desired therapeutic results. These include, for instance, breast reconstruction, face rejuvenation, or the treatment of chronic or nonhealing wounds.^27–29 In these cases, cryopreservation of adipose tissue would represent an attractive approach, because it could avoid repeated liposuctions and, thus, minimize donor site morbidity, patient discomfort, and eventually health care costs. Accordingly, during the last years, an increasing number of studies have investigated the effects of cryopreservation on adipose tissue and several protocols have been established to preserve its composition and biological properties with a wide range of results, and to some extent, contradictory findings. Moreover, notable advancements in cryopreservation technologies, such as vitrification of hydrogel-embedded tissues, nanoparticle-assisted warming methods, and enrichment of cryopreserved tissue with anti-oxidant compounds, have been proposed.^30–34

The present review aims to provide a comprehensive overview of adipose tissue cryopreservation with a focus on influencing factors that crucially determine the outcome of cryopreservation. These include the use of cryoprotectants, the cooling and warming rate, the storage temperature, and the supplementation or enrichment of cryopreserved fat grafts. Moreover, this review explores the potential characteristics of an ideal fat cryopreservation protocol, which, however, may not be applicable across all cases for several reasons. Finally, this review highlights emerging cryopreservation techniques and offers recommendations for future research directions.

Material and Methods

Review question and search strategy

The currently existing literature was reviewed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (http://www.prisma-statement.org) using the PubMed/MEDLINE database. The following search algorithm was applied: (fat OR adipose tissue OR fat grafting OR fat transplantation OR lipoaspirate OR macrofat OR microfat OR nanofat) and (cryopreservation). The literature search encompassed studies published up to November 2024. Bibliographic references in identified articles were also cross-checked for additional relevant publications not captured by the initial database search. No publication date restrictions were applied. Duplicate records were removed.

Selection criteria

Inclusion and exclusion criteria were defined before undertaking the search. Eligibility criteria encompassed articles written in English with original preclinical or clinical data on cryopreservation of unprocessed or mechanically processed adipose tissue (macrofat, microfat, and nanofat). In contrast, studies on cryopreservation of ASCs, SVF, microvascular fragments, or other isolated components of adipose tissue were not included. To be eligible, the articles needed to report data on the applied cryopreservation protocol, at least the storage temperature. The following study types were excluded: abstracts, letters, editorials, comments, case reports, and reviews. Two investigators (F.B. and E.L.) screened and selected all the studies independently, yet in a nonblinded way, relying on titles and abstracts. Subsequently, the full texts were examined in detail to determine eligibility. A third reviewer (M.W.L.) was consulted in case of discrepancies while interpreting the literature.

Data extraction

Following data were collected by one author (F.B.) from each identified article and organized in a table: reference and country of publication, type of study, donor and recipient species, processing technique, rate of cooling, storage temperature (°C), type of cryoprotective agent (CPA), storage duration (days), rate of warming, supplementation, and main message (Table 1).

Table 1.

Overview of All Included Studies

Reference, country	Type of study	Donor species	Recipient species	Processing technique	Rate of cooling	Storage temperature (°C)	Type of CPA	Storage duration (days)	Rate of warming	Supplementation	Quality assessment	Main message
Almeida et al., 2014³⁵; Brazil	Preclinical	Rat	—	—	Fast	4 –196	10% DMSO + FBS	1, 7	Slow	—	Good	Cryopreservation without CPA preserves fat performance better than with DMSO
Atik et al., 2006³⁶; Turkey	Preclinical	Murine	Mouse	—	Fast	−35	Glycerol	180	Slow	—	Fair	Cryopreservation in liquid nitrogen and subsequent storage at −35°C preserves fat performance
Badowski et al., 2019³⁷; USA	Preclinical	Human	—	—	Slow	−180	5% DMSO + 1% dextran-40 + 1% HSA	13–1320	Fast	—	Fair	Cryopreservation preserves fat performance
Bae et al., 2015³⁸; Korea	Preclinical	Human	Mouse	Centrifugation	Fast	−20	—	240	Fast	ASCs SVF	Fair	Cryopreservation with ASCs enrichment preserves fat performance better than with SVF enrichment or without enrichment
Bae et al., 2016³⁹; Korea	Preclinical	Human	Mouse	Centrifugation	Fast	−20	—	—	Fast	SPC 1, 3, 5, 10, 15 µM	Fair	Cryopreservation with SPC enrichment preserves fat performance better than without enrichment
Bagge et al., 2023⁴⁰; Denmark	Preclinical	Human	—	Microfragmentation	Slow	−80	FBS + 10% DMSO	46–150	Fast	—	Good	Cryopreservation of lipogems at −80°C with 10% DMSO + FBS preserves fat performance
Butterwick et al., 2006⁴¹; USA	Clinical	Human	Human	Centrifugation	Fast	−40	—	17	Slow	—	Fair	Short-term cryopreservation preserves fat performance in the clinical setting
Chaput et al., 2013⁴²; France	Preclinical	Human	Mouse	Centrifugation	Slow	−80 –196	HSA HES 2.5% DMSO + HSA 2.5% DMSO + HES 5% DMSO + HSA 5% DMSO + HES	7, 30	Fast	—	Fair	Cryopreservation with 5% DMSO+HES at −80°C preserves fat performance better than with other CPAs or at −196°C
Choudhery et al., 2013⁴³; Pakistan	Preclinical	Human	—	—	Slow	−196	70% basal medium + 20% serum + 20% DMSO	7	Fast	—	Fair	Short-term cryopreservation with 70% basal medium + 20% serum + 20% DMSO, slow freezing, and fast thawing at −180°C preserves fat performance
Conti et al., 2015⁴⁴; Italy	Preclinical	Human	Mouse	—	Fast	−196	10% glycerol + 0,1% HSA + 0.1 M sucrose	15	Fast	—	Fair	Short-term cryopreservation at −196°C with glycerol preserves stem cell potential
Cui et al., 2007⁴⁵; USA	Preclinical	Human	Mouse	Centrifugation	Slow	−196	0.2 M DMSO + 0.1 M trehalose 0.5 M DMSO + 0.2 M trehalose 0.25 M trehalose 0.5 M trehalose 1.0 M DMSO 1.5 M DMSO	<1 (1 h), 7	Slow-fast	—	Fair	Cryopreservation with 0.5 M DMSO + 0.2 M trehalose, slow freezing, and thawing protocol preserves fat performance better than with other CPAs or without CPA
Cui et al., 2009⁴⁶; USA	Preclinical	Human	—	Centrifugation	Slow	−196	Trehalose 0.2 mol/L Trehalose 0.25 mol/L Trehalose 0.3 mol/L Trehalose 0.35 mol/L Trehalose 0.4 mol/L Trehalose 0.5 mol/L Trehalose 0.75 mol/L	<1 (20 min)	Slow-fast	—	Fair	Cryopreservation with trehalose alone preserves fat performance, especially at a concentration of 0.35 mol/L
Cui et al., 2010⁴⁷; USA	Preclinical	Human	Mouse	Centrifugation	Slow	−196	0.5 M DMSO + 0.2 M trehalose 0.35 M trehalose	<1 (20 min)	Slow-fast	—	Fair	Cryopreservation with 0.35 M trehalose or with 5 M DMSO + 0.2 M trehalose are equivalent in the preservation of fat performance
Devitt et al., 2015⁴⁸; USA	Preclinical	Human	—	—	—	−70	—	2–1159	—	—	Fair	Long-term cryopreservation at −70°C preserves fat performance
Erdim et al., 2009⁴⁹; Turkey	Preclinical	Human	—	—	—	4 –20 –80	—	14	Slow	—	Fair	Cryopreservation at 4°C preserves fat performance better than other temperatures
Erol et al., 2013⁵⁰; Turkey	Clinical	Human	Human	Centrifugation	Fast	−80	—	90, 180, 365	Slow	—	Fair	Long term cryopreservation at −80°C without CPA preserves fat performance in the clinical setting
Favaretto et al., 2023⁵¹; Italy	Preclinical	Human	—	Filtration	Slow	−160 –80	10% DMSO + 2% albumin	30, 60, 90, 420, 1080	Fast	—	Good	Cryopreservation at −80°C and −160°C with CPA preserves fat performance
Feng et al, 2019⁵²; China	Preclinical	Human	Mouse	Centrifugation Centrifugation, filtration and emulsification (SVF gel)	Fast	−20	—	30, 90	Fast	—	Fair	Short-term cryopreservation of SVF gel at −20°C without CPA preserves fat performance better than cryopreservation of Coleman fat
Galhuber et al., 2021⁵³; Austria	Preclinical	Mouse	—	—	Fast	−80 –196	—	—	Slow	—	Fair	Cryopreservation preserves fat performance
Grewal et al., 2009⁵⁴; USA	Preclinical	Human	Rat	—	Fast	−20	50% DMEM + 40% FBS + 10% DMSO 10% DMEM + 80% FBS + 10% DMSO 80% FBS + 20% DMSO 30% sucrose in PBS 80% glycerol 100% DMSO	1, 7, 14, 56	—	ASCs	Fair	Cryopreservation with 30% sucrose in PBS preserves fat performance better than with other CPAs or without CPA Cryopreservation with ASCs enrichment preserves fat performance
Gu et al., 2020⁵⁵; China	Preclinical	Human	Mouse	—	Slow	−80	60% CS + 25% DMEM + 15% DMSO 30% CS + 55% DMEM + 15% DMSO	180	Fast	—	Fair	Cryopreservation with 60% CS + 25% DMEM + 15% DMSO preserves fat performance better than with 30% CS + 55% DMEM + 15% DMSO
Ha et al., 2015⁵⁶; Korea	Preclinical	Human	Mouse	Centrifugation	—	−20	—	30, 60	—	ASCs	Fair	Cryopreservation for 30 days with ASC enrichment preserves fat performance better for long-term periods or better than without ASCs
Hwang et al., 2015⁵⁷; Korea	Preclinical	Human	—	Centrifugation	—	−20	—	—	Slow Slow-fast Fast	—	Good	Cryopreservation followed by fast thawing preserves fat performance better than slow or slow-fast thawing
Jiang et al., 2023⁵⁸; China	Preclinical	Human	Mouse	—	Slow	−196	FBS + 10% DMSO	28	Fast	Exosomes	Fair	Cryopreservation with ASC exosome enrichment preserves fat performance better than without exosome enrichment
Kim et al., 2020⁵⁹; South Korea	Preclinical	Human	—	—	Slow	4 –20 –80 –196	—	30, 60, 120, 240, 360	Fast	—	Good	Cryopreservation at −80°C preserves fat performance better than at other temperatures
Ko et al., 2011⁶⁰; Korea	Preclinical	Human	Mouse	Centrifugation	Fast	−70	—	56	—	ASCs	Fair	Cryopreservation with ASC enhancement preserves fat performance
Koçak et al., 2023²⁸; Turkey	Preclinical	Human	—	—	—	−80	Adi-frosty with 100% isopropanol 90% FBS + 10% DMSO	14	—	—	Fair	Cryopreservation in adi-frosty with 100% isopropanol preserves fat performance better than with 90% FBS + 10% DMSO or without CPA
Lee et al., 2010⁶¹; Korea	Preclinical	Human	—	—	—	−20 –80	DMEM + 10% FBS + 10% DMSO	7, 30, 60, 90, 365	Fast	—	Fair	Cryopreservation with DMEM + 10% FBS + 10% DMSO at −80°C preserves fat performance
Li et al., 2012⁶²; Taiwan	Preclinical	Human	Mouse	Centrifugation	Slow	−20 –80 –196	HES	2, 7, 14, 28, 240	Fast	—	Fair	Cryopreservation with or without CPA and at different temperatures reduces fat performance
Lidagoster et al., 2000⁶³; USA	Preclinical	Rat	Rat	—	—	1 –16	—	7, 14	—	—	Fair	Short-term cryopreservation reduces fat performance
Limido et al., 2024 b²⁹; Germany	Preclinical	Mouse	Mouse	Emulsification and filtration (nanofat)	Fast Fast Slow	−20 −80	—	7	Fast	—	Fair	Short-term cryopreservation of nanofat at −20°C without CPA preserves fat performance better than at other temperatures
Lin et al., 2022⁶⁴; Taiwan	Preclinical	Human	Mouse	Centrifugation	—	−80	—	90, 120, 180, 300, 360, 720	—	ASCs	Fair	Cryopreservation with ASC enrichment preserves fat performance better than without ASC enrichment
Ma et al., 2018⁶⁵; Taiwan	Clinical	Human	Human	Centrifugation	—	−80	—	90	Fast	—	Fair	Cryopreservation at −80°C preserves clinical fat performance
MacRae et al., 2004⁶⁶; USA	Preclinical	Human	—	—	Fast Fast	−20 –196	—	2, 4, 5, 8	—	—	Fair	Short-term cryopreservation preserves fat performance better than simple incubation
Mashiko et al., 2018⁶⁷; Japan	Preclinical	Human	Mouse	—	Fast Slow	−80 –196	ACSelerate-CP American CryoStem	14	Fast	—	Fair	Cryopreservation reduces fat performance regardless of the protocol used
Massiah et al., 2021⁶⁸; Italy	Preclinical	Human	—	Centrifugation	Slow	−196	5% DMSO 10% DMSO + 20% HSA	90	Fast	—	Good	Cryopreservation without CPA preserves fat performance comparably to cryopreservation with CPA
Matsumoto et al., 2007⁶⁹; Japan	Preclinical	Human	—	—	Slow	4 –80	Cryopreservation medium (Cell Banker BLC-1; ZENOAQ, Fukushima, Japan)	1, 2, 3, 30	Fast	—	Fair	Cryopreservation reduces fat performance
Moscatello et al., 2005⁷⁰; USA	Preclinical	Human	—	Centrifugation	Fast Slow	−20 –80 –196	MCDB medium 10% DMSO 7.5% PVP + 7.5% DMSO 10% glycerol 10% glycerol + 10% FBS	48–244	Fast	—	Fair	Cryopreservation with CPA, slow freezing, and storage at −196°C preserves fat performance better than without CPA with fast freezing at lower temperatures
Ohashi et al., 2018⁷¹; Japan	Clinical	Human	Human	Centrifugation	Slow	−196	ACSelerate-CP American CryoStem	—	Fast	—	Fair	Cryopreservation slightly reduces clinical fat performance, but the procedure is complication-free
Ohashi, 2020⁷²; Japan	Clinical	Human	Human	Centrifugation	Slow	−196	ACSelerate-CP American CryoStem	—	Fast	—	Fair	Cryopreservation slightly reduces clinical fat performance, but the procedure is complication-free
Ohta et al., 2018⁷³; Japan	Preclinical	Rat	Rat	—	Fast	−80	TC-Protecter, DS Pharma Biomedical, Osaka, Japan	14, 30, 84, 168	Fast	—	Fair	Short-term cryopreservation preserves fat performance better than long-term cryopreservation
Pu et al., 2004⁷⁰; USA	Preclinical	Human	—	Centrifugation	Slow	−196	0.5 M DMSO + 0.2 M trehalose	<1 (20 min)	Slow-fast	—	Good	Cryopreservation with 0.5 M DMSO + 0.2 M trehalose preserves fat performance better than without CPA
Pu et al., 2005⁷⁴; USA	Preclinical	Human	—	Centrifugation	Slow	−196	0.25 M trehalose	<1 (20 min)	Slow-fast	—	Good	Cryopreservation with 0.25 M trehalose preserves fat performance better than without CPA
Pu et al., 2006a⁷⁵; USA	Preclinical	Human	Mouse	Centrifugation	Slow	−196	0.5 M DMSO + 0.2 M trehalose	<1 (20 min)	Slow-fast	—	Fair	Cryopreservation with 0.5 M DMSO + 0.2 M trehalose preserves fat performance better than without CPA
Pu et al., 2006 b⁷⁶; USA	Preclinical	Human	—	Centrifugation	Slow	−196	0.5 M DMSO + 0.2 M trehalose	<1 (20 min)	Slow-fast	—	Good	Cryopreservation with 0.5 M DMSO + 0.2 M trehalose preserves fat performance
Pu et al., 2010⁷⁷; USA	Preclinical	Human	—	Centrifugation	Slow	−196	0.5 M DMSO + 0.2 M trehalose	<1 (20 min)	Slow-fast	—	Good	Cryopreservation with 0.5 M DMSO + 0.2 M trehalose preserves fat performance
Quan et al., 2022⁷⁸; China	Clinical	Human	Human	Centrifugation, filtration and emulsification (SVF gel)	Fast	−20	—	90	Fast	—	Fair	Cryopreservation of SVF gel at −20°C without CPA preserves clinical fat performance
Roato et al., 2016⁷⁹; Italy	Preclinical	Human	—	Centrifugation	Slow	−20 –80 –196	FBS + 10% DMSO FBS + 0.35 M trehalose	—	Fast	—	Fair	Cryopreservation with DMSO at −80°C or −196°C preserves fat performance better than with trehalose or at lower temperatures
Rusconi et al., 2024⁸⁰; Switzerland	Preclinical	Human	—	—	Slow	−150	40% Cryo-Sure DEX40 + 40% MEM alpha no phenol red + 20% HSA	7–90	Fast	—	Fair	Cryopreservation with 40% Cryo-Sure DEX40 + 40% MEM alpha no phenol red + 20% HSA preserves fat performance better than without CPA
Shaik et al., 2020⁸¹; USA	Preclinical	Human	—	—	Slow	−20 –80	10% DMSO + 35% BSA + DMEM 2% DMSO + 43% BSA + DMEM 10% DMSO + 35% LS + DMEM 2% DMSO + 6% HSA + DMEM 40% LS + 10% PVP + DMEM	30	Fast	—	Fair	Cryopreservation with CPA at −80°C preserves fat performance better than at other temperatures
Shoshani et al., 2001⁸²; Israel	Preclinical	Human	Mouse	Centrifugation	Fast	−18	—	14	Slow	—	Fair	Short-term simple freezing preserves fat performance
Tao et al., 2022⁸³; China	Preclinical	Human	Mouse	Centrifugation, filtration and emulsification (SVF gel)	Fast	−20	—	5, 15, 45	Fast	—	Fair	Short-term cryopreservation of SVF gel at −20°C without CPA preserves fat performance better than long-term cryopreservation
Ullmann et al., 2004⁸⁴; Israel	Preclinical	Human	Mouse	Centrifugation	Fast	−18	—	210	Slow	—	Fair	Long-term simple freezing reduces fat performance
Ventura et al., 2024⁸⁵; Italy	Preclinical	Human	—	Fractionation	Slow	−195	0.5 M DMSO + 0.2 M trehalose	30, 90, 180, 360, 540	Fast	—	Fair	Cryopreservation of fractionated adipose tissue at −195°C with CPA, slow freezing, and fast thawing preserves fat performance
Villaverde-Domenech et al., 2021⁸⁶; Spain	Preclinical	Human	Mouse	Centrifugation	Slow	−20 –80	Trehalose HES	30, 60	Fast	—	Fair	Cryopreservation preserves fat performance equally at −20°C or −80°C, with trehalose or HES
Wang et al., 2013⁸⁷; USA	Preclinical	Human	—	—	Slow	−80	NHEM + 10% FBS + 10% DMSO + 1% penicillin-streptomycin	30	Fast	—	Fair	Cryopreservation with NHEM + 10% FBS + 10% DMSO + 1% penicillin-streptomycin preserves fat performance better than without CPA
Zanata et al., 2017⁸⁸; Brazil	Preclinical	Human	Mouse	—	Slow	−196	70% FBS + 20% DMSO + 10% stromal medium	120–180	Fast	—	Fair	Cryopreservation reduces fat performance
Zhang et al., 2022⁸⁹; China	Preclinical	Human	Mouse	Centrifugation	Slow	−196	60% glycerol + PBS solutions 70% glycerol + PBS solutions 80% glycerol + PBS solutions 90% glycerol + PBS solutions 100% pure glycerol 0.25 mol/L trehalose + PBS solution 90% FBS + 10% DMSO	30	Fast	—	Fair	Cryopreservation with 70% glycerol preserves fat performance better than with other CPAs
Zheng et al., 2019⁹⁰; China	Preclinical	Human	Mouse	Centrifugation	Slow	4 –20 –80 –196	—	90	Fast	SVF	Fair	Cryopreservation without CPA is possible, cryopreservation at −80°C for 90 days with SVF enrichment preserves fat performance better than at other temperatures or without SVF enrichment

Where available, the type of study, donor species, recipient species, processing technique, rate of cooling, storage temperature (°C), type of CPA, storage duration (days), rate of thawing, supplementation, and main message are provided.

ASC, adipose-derived stem cell; BSA, bovine serum albumin; CPA, cryoprotectant agent; CS, calf serum; DEX40, Dextran 40; DMEM, Dulbecco’s modified Eagle’s medium; DMSO, dimethylsulfoxide; FBS, fetal bovine serum; HSA, human serum albumin; HES, hydroxyethyl starch; LS, lipoaspirate saline; MEM, minimum essential medium; NHEM, nonhematopoietic expansion medium; PBS, phosphate-buffered saline; PVP, polyvinylpyrrolidone; SPC, sphingosylphosphorylcholine; SVF, stromal vascular fraction.

Risk of bias assessment

Risk of bias assessment of included articles was conducted by a single author (F.B.). Preclinical in vivo studies were evaluated using the SYRCLE’s risk of bias tool for animal studies.³⁷ For clinical studies, the NIH tool (https://www.nhlbi.nih.gov/health-topics/study-quality-assessment-tools) was utilized. In case of in vitro studies, we adapted the NIH tool by excluding questions not applicable to in vitro analyses. The final judgment on study quality (i.e., poor/fair/good quality) is presented in Table 1.

Results and Discussion

The initial database search in PubMed/MEDLINE identified a total of 678 articles published up to November 2024. An additional record was identified through a review of the bibliographies of these articles. After title and abstract screening, 67 studies were selected for full-text review. Of these, 8 studies did not meet the inclusion criteria and were therefore excluded. Consequently, 59 articles were included in the analysis. The flowchart of the study selection process is presented in Figure 2, whereas the details of all included studies are summarized in Table 1.

FIG. 2.

Flowchart displaying the study selection process of this systematic review.

In the following subsections, the main results from the included 59 articles are presented and discussed with a focus on different aspects of the cryopreservation procedure. These include the use of cryoprotectants, the cooling and warming rate, the storage temperature, and the supplementation or enrichment of cryopreserved fat.

Cooling rate

Exposure to subzero temperatures can be detrimental to cells and tissues. This damage results mostly from uncontrolled transitions between normothermic and hypothermic conditions.⁴⁰ Depending on the cell or tissue type, there is a great diversity in cryobiological responses during the cooling and warming cycle. Mazur⁴² described, for the first time, the cellular response to cooling. During the cooling process, water inside the cells initially remains unfrozen and supercooled, because the plasma membrane prevents intracellular ice crystal formation while nucleation occurs extracellularly.⁴³ Nucleation represents the initial step in ice formation,⁴⁰ followed by the growth of ice crystals from these nucleation sites. Subsequently, as water is removed from the extracellular fluid to form ice crystals, the extracellular solute concentration increases. Due to the resultant osmotic gradient across the cell membranes, water flows out of the cells, resulting in cell dehydration and increased intracellular solute respectively electrolyte concentrations. This increased solute concentration depresses the intracellular freezing point, thereby preventing intracellular ice formation.⁴⁰

If the cooling rate is too slow, excessive dehydration occurs, resulting in osmotic stress or membrane damage due to extracellular ice formation (Fig. 3). On the contrary, excessively rapid cooling prevents adequate cellular dehydration, resulting in ice formation of the residual intracellular water, which can cause rupture of the cell membrane.⁴³ To minimize the extent of ice-related damage, cryopreservation protocols usually employ CPAs⁵¹ that reduce the concentration of electrolytes and also ice formation,⁵⁵ as detailed in the following section.

FIG. 3.

Biophysical mechanisms during cooling depending on the cooling rate and the use of CPAs. At normothermic conditions, water exchange between the intra- and extracellular space is balanced (A). During cooling, extracellular nucleation and ice crystal growth occur. As extracellular water becomes part of the ice crystals, extracellular solute concentration increases, resulting in osmotic dehydration of cells. A too slow cooling rate causes excessive dehydration leading to osmotic stress and membrane damage (B). Conversely, cooling too fast limits water efflux from the cells, causing intracellular ice formation, which can rupture the cell membrane (D). These injuries can be prevented by slow cooling at a controlled rate using CPAs to allow water efflux from the cells without osmotic stress and intracellular ice formation (C). Another cryopreservation strategy is vitrification, which applies a high cooling rate and CPA concentration to suppress ice formation, resulting in a glass-like solidification of cells or tissues (E).

To avoid cryoinjury, cells can be cooled slowly in a controlled way with a gradual cooling rate of −1°C per minute. This cooling rate is slow enough to reduce ice crystal formation intracellularly while minimizing the solution effects, thereby enabling the use of low concentrations of CPAs (Fig. 3).⁷¹ This so-called “slow cooling” is the most traditional cryogenic preservation procedure that is primarily used for single-cell suspensions.⁷² In contrast, slow cooling of adipose tissue is more challenging, because it is necessary to ensure that CPA diffusion and removal occurs in a harmonious way and to guarantee a uniform freezing of the tissue structure.^72,73,88

An alternative well-established approach to avoid cryoinjury during cooling is vitrification.⁵² Vitrification is defined as the solidification of cells or tissues at the so-called “glass transition temperature” around −80°C and −130°C with no ice crystal formation.⁴⁰ In case of very small biological samples, vitrification can be achieved without CPAs using an extremely fast cooling rate.⁷⁸ However, this extremely fast cooling rate cannot be practically achieved for larger living systems.⁴⁰ For this purpose, vitrification requires very high concentrations of CPAs to decrease the critical cooling rate to practical levels, allowing sample solidification before ice formation occurs (Fig. 3).^83,91

Of the 59 studies included, 33 involved study groups using a slow cooling rate with 78% of studies (26 out of 33) reporting favorable outcomes in maintaining tissue survival and structure. On the contrary, 20 studies investigated cryopreservation study groups with a fast cooling rate and 90% (18 out of 20) reported the success of this approach. However, the vitrification technique was not explicitly mentioned in any of these 20 studies. In fact, in many cases the adipose tissue was simply cooled to subzero temperatures with a fast cooling rate without reaching the “glass transition temperature.” In contrast, other studies applied a true vitrification protocol, although it was not defined in this way. Moreover, several other reports failed to provide information regarding the cooling rate.^{28,36,54,61,70,74,75,80,87} In addition, three studies specifically compared study groups with slow and fast cooling rates, but the results were divergent and often not completely evaluable due to the presence of confounding variables (e.g., additional use of CPAs, storage temperature, etc.). Specifically, Mashiko et al.³⁵ compared fast cryopreservation of adipose tissue at −80°C without CPAs and cryopreservation with slow cooling to −196°C in a cryopreservation medium. In both cases, the preserved adipose tissue was unsuitable for clinical use due to a low cell viability. Moscatello et al.⁶² showed that a slow cooling approach with a CPA maintains cell viability, whereas fast cooling of fat at −20°C markedly induces cell death. Limido et al.²⁹ reported that slow cooling to −80°C reduces the degree of apoptotic cell death within nanofat in comparison with other protocols, but they also found that fast cooling at −20°C preserves cells from apoptosis.

These findings do not show a clear indication for the use of either slow cooling, vitrification, or fast cooling at temperatures below the “glass transition temperature” in case of adipose tissue and its derivatives. Of note, these techniques have often been reported confusingly. Vitrification has sometimes been used without defining it properly, and sometimes the cooling rate is not specified at all. Moreover, the results of individual studies are often divergent from each other. In addition, as mentioned previously, the primary challenge of cooling tissues or organs when compared with cell suspensions is that cooling does not occur homogeneously. In fact, tissue samples usually nucleate firstly at the border zones and then in the center. As a result, the well-established techniques of slow cooling and vitrification are not completely applicable for tissues. Recently, innovative approaches have been proposed to address nonhomogeneous cooling, for instance, directional freezing and directional vitrification. Directional freezing is a method that uses a temperature gradient to reduce progressive ice growth and to allow uniform cooling by forming ice lamellae that trap cells and minimize mechanical damage (Fig. 4).⁴⁰ This technique has already been tested for various organs and tissues^67,68,76 but has not yet been applied to adipose tissue. Directional vitrification is based on the same principle but involves a very rapid cooling rate and has been explored less extensively.⁴⁰ Moreover, Pu et al.³⁰ developed a microfluid-based approach for tissue vitrification, encapsulating mesenchymal stroma cells in gelatin methacrylate hydrogel. This technique provides a practical solution for the cryopreservation of multicellular tissues, while also reducing the need for CPAs.³⁰ Taken together, these emerging approaches seem to be promising for the cryopreservation of fat and its derivatives, warranting further investigation in future studies.

FIG. 4.

Principle of directional freezing. Cold is applied to one side of the sample causing a temperature gradient. Uniform ice lamellae form progressively along this gradient with heat being conducted through the unfrozen part of the sample.

Cryoprotectant

CPAs are used to maintain viability when cooling cells, tissues or organs.⁵⁵ CPAs can stabilize the cell membrane, minimize osmotic stress, and suppress intra- and extracellular ice crystal formation by reducing the cellular water content and increasing the total concentration of all solutes.⁷² After warming and before transplantation of cryopreserved cells, tissues or organs, CPAs are usually discarded to avoid adverse reactions, such as nausea, vomiting, and abdominal cramps as well as cardiovascular, respiratory, and neurological symptoms.⁷²

CPAs can be classified into low-molecular-weight permeating CPAs, high-molecular-weight nonpermeating CPAs, and nonpermeating sugar.⁷² Permeating CPAs penetrate the cell membrane and remove the water from the cytoplasm, which inhibits the formation of intracellular ice crystals. However, these CPAs may adversely affect cells by inducing toxicity and osmotic stress, particularly when they penetrate the cell membrane at a rate that differs from the efflux of water. In fact, faster entry leads to the swelling of the cytoplasm, which may result in membrane disruption. On the contrary, slower entry increases extracellular osmotic forces that damage the cell by excessive shrinkage.⁷² The membrane crossing rate depends on the type of cryoprotectant, its concentration, and the surrounding microenvironment, such as the ambient temperature. High CPA concentrations were found to be toxic to cells, especially at high temperatures, which correlate to an increased risk of osmotic shock.^45,46

Nonpermeating CPAs are often used in combination with permeating CPAs to minimize cell volume changes and accelerate dehydration during cooling.^72,77 Sugars, such as sucrose and trehalose, along with potentially high-molecular-weight polymers contribute to cellular protection by forming a viscous coating on the cell surface. This layer helps to regulate osmotic pressure between intra- and extracellular environments while stabilizing both the cell membrane and associated proteins.⁷⁷ Because sugars do not penetrate the cell membrane, they are less toxic. However, they do not prevent intracellular ice crystal formation. Hence, they are considered less effective when used alone.⁷²

In the last decade, many types of CPAs have been investigated for the cryopreservation of adipose tissue.^{47,49,63,85,92–97} Furthermore, studies have evaluated cryopreservation protocols excluding the use of CPAs to avoid their associated toxicity.^48,49,56,57 This is particularly the case for the cryopreservation of fat derivatives, such as nanofat, where it is technically not possible to effectively separate CPAs from nanofat without affecting its original composition and consistency.²⁹ Of the 59 studies included in this systematic review, 36 involved study groups using a CPA with 80% of the studies (29 out of 36) reporting favorable outcomes in maintaining tissue performance. On the contrary, 37 studies included study groups with CPA-free cryopreservation and in 51% of the cases (19 out of 37) the cryopreserved tissue retained a similar structure and biological activity as fresh adipose tissue. Fourteen of these studies specifically compared the cryopreservation study groups with and without CPAs. In nine of these studies, the use of CPAs yielded better results,^{28,59,62,64–66,69,75,90} whereas two studies showed improved outcomes with CPA-free protocols^44,79 and three studies reported comparable results for the two approaches.^35,53,89 Thus, the use of CPAs still remains a controversial topic.

Dimethylsulfoxide (DMSO) is a permeating CPA that has been widely used for cryopreservation of adipose tissue with good results in the majority of the herein included studies (21 out of 26). Given its cytotoxicity and the practical challenges associated with its removal from cryopreserved tissue in clinical settings, extensive research has been performed to identify more suitable CPAs. Several studies reported that trehalose together with a lower dose of DMSO or even alone results in comparable cryoprotection.^{41,58,64,65,69,81,82,84,86} This nonpermeating sugar plays a crucial role in stabilizing the cell membrane during dehydration due to its capability of high water retention. Moreover, trehalose facilitates ion transport across the cell membrane, decreases the melting point of membrane lipids and improves the stability of proteins.⁵⁰

Taken together, most studies report favorable outcomes in terms of tissue survival and histological structure by using CPAs, particularly DMSO. Therefore, it may be concluded that cryopreservation protocols for adipose tissue should include DMSO. However, it should be considered that in some specific cases, this cannot be realized easily. Considering for instance nanofat, a proper separation of the CPA from nanofat is impossible without markedly changing the composition and consistency of this fat derivative. Accordingly, further research is needed to establish novel DMSO- or CPA-free protocols that do not affect the quality of distinct types of cryopreserved adipose tissue grafts. Nonpermeating CPAs, such as trehalose, represent a good alternative. Moreover, Zhou et al.⁶⁰ recently reported a novel stem cell cryopreservation platform based on an antifreezing polyvinylpyrrolidone/gellan gum/gelatin scaffold together with an L-proline-assisted cell predehydration strategy. This platform has been proven to be capable of inhibiting ice formation without using any toxic CPAs, which makes traditional washing processes needless.⁶⁰ Furthermore, even in cases in which a permeating CPA can be used, the toxicity of the compound remains a major challenge. Importantly, the toxic effect of CPAs increases with temperature and concentration. Therefore, an alternative strategy could be the stepwise CPA delivery and removal during cooling and warming, reducing toxicity and osmotic stress.^40,52 However, this method requires further development.

Storage temperature

The storage temperature is an additional variable of utmost importance for a proper cryopreservation. It has been demonstrated that cooling to hypothermic temperatures of ∼4°C leads to the preservation of cells or tissues.⁷² In fact, such temperatures slow down energy-dependent processes, such as protein synthesis, transport, and cell cycle progression, without damaging neither cells nor extracellular matrix components due to ice crystal formation and changes in solute concentration. However, storage at hypothermic temperatures has also been shown to have detrimental effects on cells, including the generation of reactive oxygen species, which occurs in direct proportion to the duration of preservation. Therefore, it can only be applied for short periods of time.⁷² Comparing cell viability of adipose tissue at different storage temperatures, Matsumoto et al.⁹⁸ observed that preservation for 24 h at 4°C shows a similar yield of ASCs as fresh tissue, but the yield drops to uncertain levels already after 2 or 3 days. Zheng et al.³⁸ reported that viable cells can be isolated from adipose tissue stored for 1 week at 4°C. Erdim et al.⁷⁴ demonstrated that the storage of adipose tissue at 4°C for 14 days preserves the viability of adipocytes. Kim et al.⁹⁹ found good cell survival at 4°C even after 28 days of cryopreservation. However, these cells already lost their ability to proliferate after 7 days.

Cryopreservation at very low temperatures is required for long-term storage. For this purpose, different cooling temperatures have been analyzed, especially −196°C (24 out of 59 studies), −80°C (23 out of 59 studies), and −20°C (19 out of 59 studies). Many studies suggest that cryopreservation at −196°C in liquid nitrogen represents a valid storage option with good preservation outcomes.^{41,58,62,64,65,69,79,81,82,84,86,89,100–105} However, this approach requires an elaborate and complex facility and is therefore associated with high costs. For this reason, it may not always be easy to implement in daily clinical practice. On the contrary, cryopreservation at −20°C or −80°C may be more accessible for surgeons in daily clinical routine. Comparing the effects of cryopreservation at these two temperatures, several studies claim that −80°C is more effective for the storage of lipoaspirates in terms of cell viability.^35,38,99,106 However, Limido et al.,²⁹ Li et al.,⁵³ and Villaverde-Domenech et al.¹⁰⁷ also reported good cell viability rates for a temperature of only −20°C.

Taken together, the ideal temperature for the cryopreservation of adipose tissue remains a matter of discussion. However, it seems rather obvious that very low temperatures are required for long-term preservation, whereas short-term preservation might effectively be done with less low temperatures. Furthermore, recent studies highlighted that protocols involving cryopreservation at −80°C or −20°C may be more easily implemented in clinical routine when compared with storage at −196°C. Moreover, these protocols have reported satisfactory results, especially when using a temperature of −80°C.

Warming rate

The cryopreserved tissue finally experiences the stress of warming, which can also result in significant cell injuries comparable with those induced by the cooling procedure. During warming, ice crystals can further grow if nucleation has occurred. This is due to the fact that both nucleation and ice crystal growth depend on the temperature but in different ways. The process of ice growth occurs mostly just below the freezing point and then it attenuates when the temperature drops. On the contrary, nucleation peaks well below the freezing point.⁴⁰ Therefore, during cooling, the sample passes the ice growth temperature but with few nuclei available. Thereafter, when it reaches the nucleation temperature, crystallization is no longer optimal. In contrast, during warming, the risk of recrystallization is higher if several nuclei have formed.⁴⁰

Nine studies included in the present review investigated study groups with slow warming at room temperature.^{44,54,74,79,103,108–111} However, most studies (35 out of 59 studies) proposed that fast warming, usually in a 37°C water bath, should be preferred. The underlying idea is that fast warming can prevent cell death by inhibiting, in case of previous intracellular ice formation, the detrimental growth of small intracellular ice crystals into larger crystals by recrystallization.⁴³ Moreover, nine studies tested study groups with slow-fast warming.^{54,58,64,65,69,81,82,84,86} This approach is characterized by an initial slow warming at room temperature for 2–5 min followed by fast warming in a 37°C water bath. Only one of these studies specifically compared study groups with slow, fast, and slow-fast warming rates, demonstrating the superiority of fast warming in terms of mitochondrial activity and adipocyte viability.⁵⁴ Eight studies did not provide information about the warming rate.^{28,36,61,70,87,90,100,112}

These findings indicate that, as it is for many other types of tissues, fast warming should also be applied for cryopreserved adipose tissue. However, as it is for cooling, fast uniform warming can be easily achieved only for single-cell suspensions. In contrast, tissues exhibit a low thermal conductivity and large volumes. Accordingly, conventional heating in a preheated water bath can cause large temperature gradients within tissues. Nonuniform heating, in turn, causes a high thermal mechanical stress that can lead tissues to crack or fracture. Recently, the new technology of inductive warming with magnetic nanoparticles has been tested to achieve a faster uniform warming of porcine arteries and aortic heart valve leaflet tissue.⁴⁰ Of interest, this approach successfully reduced thermally-induced mechanical stress and, hence, fractures of the analyzed tissues. Therefore, this approach may be also promising for the cryopreservation of adipose tissue, which should be analyzed in future studies.

Supplementation/enrichment of cryopreserved fat grafts

To further improve the in situ performance of cryopreserved fat, the possibility of supplementing frozen adipose tissue with cells or other biological active compounds has recently been investigated in preclinical studies. In fact, since the enrichment with ASCs, exosomes or SVF has already been shown to improve the vascularization and survival rate of transplanted fresh adipose tissue,^36,113,114 these components have also been proposed to be beneficial for cryopreserved adipose tissue.

Five studies tested the supplementation of ASCs to frozen adipose tissue grafts.^{36,87,90,112,113} ASCs improved the structure and viability of the cryopreserved tissue by reducing necrosis and fibrosis.^{87,90,112,113} In addition, ASC-enriched cryopreserved adipose tissue grafts exhibited an enhanced vascularization, as indicated by an increased concentration of vascular endothelial growth factor (VEGF) and expression of CD31.^36,113 In line with this finding, it is well known that ASCs promote blood vessel formation and adipocyte differentiation while preventing apoptosis by releasing VEGF, hepatocyte growth factor and insulin-like growth factor-1 and by differentiating into endothelial or adipose cells.^39,115–121

The SVF is a fat derivative that is obtained by enzymatic digestion of adipose tissue and contains a mixture of single cells, including endothelial cells, smooth muscle cells, pericytes, fibroblasts and ASCs, with a high regenerative potential.¹²² Therefore, its use has been considered to enhance the viability of frozen fat. Zheng at al.³⁸ demonstrated that the SVF can increase the survival of frozen fat grafts by promoting vascularization and adipocyte differentiation. In contrast, Bae et al.¹¹³ reported no effect of the SVF on the survival and quality of transplanted frozen fat.

Moreover, Jiang et al.¹⁰⁵ studied the effects of ASC-derived exosomes on cryopreserved adipose tissue activity after engrafting. ASC-derived exosomes are active vesicles that are secreted by ASCs and transport various biological components, such as lipids, proteins, and microRNAs.^123–125 ASC-derived exosomes are not only more convenient to be stored when compared with ASCs,¹²⁶ but they have also been shown to promote angiogenesis and eventually increase the retention rate of grafted fat.^{19,114,127–130} However, in the study by Jiang et al.,¹⁰⁵ ASC-derived exosomes only improved the survival rate of cryopreserved fat grafts in the short term due to a reduction of fibrosis and oil cyst formation as well as an increased M2 macrophage polarization within the first 4 weeks after transplantation. Thereafter, the positive effects of these exosomes were no longer detectable. Hence, these findings indicate that the utility of using ASC-derived exosomes to treat cryopreserved fat grafts seems to be limited so far, and, thus, requires further optimization in the future.

In addition, lysophospholipid sphingosylphosphorylcholine (SPC) has been investigated as a promising supplement for cryopreserved adipose tissue.¹³¹ SPC is known to stimulate DNA synthesis and proliferation of ASCs.^132–134 Furthermore, SPC exerts a strong mitogenic effect on several cell types, including endothelial cells.¹³⁵ Of interest, Bae et al.¹³¹ reported that SPC may exert favorable effects on grafted cryopreserved human adipose tissue, probably due to the upregulation of gene transcription associated with angiogenesis.

Taken together, all herein presented enrichment strategies supplement frozen adipose tissue with cells or other biologically active compounds exerting regenerative and proangiogenic effects. To date, the supplementation of ASCs seems to be the most promising strategy that has been tested most frequently with favorable results in terms of preserving the viability of frozen fat to be grafted. Of interest, other enrichment strategies have also been proposed for tissues other than fat, i.e., the supplementation of N-acetylcysteine, epimedium polysaccharide, or pyrroloquinoline quinone.^32–34 These new strategies should also be tested in the context of fat cryopreservation in the future.

Conclusions and Future Perspectives

Fat grafts are increasingly used in plastic, reconstructive, and esthetic surgery, not only as fillers or volumizers but also as regenerative autologous components. Cryopreservation is a promising technique for the storage of adipose tissue. In fact, it may allow multiple sessions of autologous fat transfer or the repeated isolation of ASCs from one single source, while avoiding repeated liposuction procedures to harvest the tissue. Hence, it can markedly contribute to minimize patient discomfort, potential complications associated with repeated surgical procedures, hospitalization, and health care costs.

Based on the comprehensive data collected throughout this systematic review (Table 2), several critical conclusions regarding the cryopreservation of adipose tissue and its implications for clinical applications can be drawn. Particularly, the herein summarized studies report, to some extent, contradictory results. This may be due to different experimental settings and, thus, possible confounding factors. For instance, the analyzed cryopreserved adipose tissues differed in terms of donor species, volume, cryopreservation time, and processing technique. Moreover, the tissue quality before cooling was not always comparable.

Table 2.

Overview of the Protocols Tested for Different Cryopreservation Variables Within Included Studies of This Systematic Review

Variable	Protocols tested
Rate of cooling	Slow cooling rate (33 studies)		Fast cooling rate (20 studies)		Not reported (9 studies)
CPAs	CPAs-dependent protocols (36 studies)			CPA-free protocols (37 studies)
Storage temperature (°C)	−196 (24 studies)		−80 (23 studies)		−20 (19 studies)
Rate of warming	Slow warming rate (9 studies)	Fast warming rate (35 studies)		Slow-fast warming rate (9 studies)		Not reported (8 studies)
Supplementation	ASCs (5 studies)	SVF (2 studies)		Exosomes (1 study)		SPS (1 study)

ASC, adipose-derived stem cell; CPA, cryoprotectant agent; SPC, sphingosylphosphorylcholine; SVF, stromal vascular fraction.

The ideal protocol for cryopreservation of fat is yet to be defined and a deeper understanding of fundamental cryobiological mechanisms and biophysical responses of adipose tissue during cooling and warming is needed. Indeed, several factors influencing fat tissue viability should be taken into consideration, including the choice of CPAs, cooling and warming rates, and storage temperatures. Each of these aspects needs further exploration starting from the results already obtained and described in this review. Specifically, it can be concluded that the cryopreservation of adipose tissue at −80°C with a CPA as well as controlled slow cooling and fast warming may currently represent the most promising protocol for further investigation, as numerous studies have applied these conditions with encouraging outcomes in maintaining tissue survival and structure. However, several aspects remain to be clarified, and it is important to note that this protocol may not be applicable in specific cases, for instance, when performing a cryopreservation of fat derivatives, such as nanofat. Moreover, in the past years, the potential of supplementing cryopreserved fat grafts with cells and/or bioactive compounds has emerged as a compelling strategy. Particularly, the incorporation of ASCs into cryopreserved adipose tissue grafts has shown promising results in terms of vascularization and eventually graft survival, indicating a beneficial avenue for future research. In addition, other innovative technologies for the cryopreservation of cells and tissues have recently been proposed, including directional freezing, the use of nonpermeating CPAs and magnetic nanoparticles-assisted inductive warming. These emerging technologies also represent promising directions for future research.

Finally, it should be considered that the implementation of adipose tissue cryopreservation into routine clinical practice represents a major translational challenge. In fact, most preclinical studies have demonstrated the feasibility of cryopreserving adipose tissue on a small scale. However, the protocols used may not be easily adaptable to larger scales, which require the establishment of adequate biobanking systems involving specialized equipment, personnel training, stringent quality control procedures, appropriate documentation, sample tracking, and compliance with regulatory standards.

Taken together, cryopreservation of adipose tissue is challenging. Nevertheless, its potential for improved patient management in reconstructive and esthetic surgical procedures is significant. Ongoing research is essential to refine protocols, enhance the viability of cryopreserved fat, and ultimately expand the clinical applications of autologous fat grafting. This review serves as a foundational step in this endeavor, encouraging further preclinical and clinical studies in this rapidly evolving field.

Authors’ Contributions

F.B.: Conceptualization, data curation, formal analysis, investigation, methodology, project administration, validation, visualization, writing-original draft preparation, writing—review and editing. E.L.: Data curation, investigation, writing—review and editing. A.W.: Writing—review and editing. Y.H.: Writing—review and editing. M.D.M.: Funding acquisition, resources, writing—review and editing. E.A.: Writing—review and editing. M.W.L.: Conceptualization, funding acquisition, project administration, resources, supervision, validation, visualization, writing—original draft preparation, writing—review and editing.

Footnotes

Acknowledgments

The authors thank Servier Medical Art for providing access to designed medical elements (), supporting the generation of graphical items in this publication.

Funding Information

There was no specific funding for this review.

Disclosure Statement

The authors have no conflicts of interest to declare.

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