Mechanical Purification of Lipofilling: The Relationship Between Cell Yield, Cell Growth, and Fat Volume Maintenance

Soft tissue defects in plastic surgery and several pathologies like as hemifacial atrophy [1], Romberg syndrome [2], ulcers [3, 4], outcomes of breast cancer [5, 6], breast hypoplasia and tuberous breast [7, 8], scars [9], and face soft tissue effects [10] have been treated with greater benefits thanks to the lipofilling. Many strategies for autologous lipofilling preparation have been reported aiming to improve long-term fat volume maintenance. Currently, several discussions seem to relate fat maintenance directly with the number of adipose-derived stem cells (ADSCs) contained in the stromal vascular fraction (SVF) of fat tissue. SVF is composed of heterogeneous cells with progenitor activity including pre-adipocytes, ADSCs, pericytes, endothelial cells, and macrophages thus providing immunomodulatory capacity for lipofilling [11]. For these reasons, several procedures of adult SVF cells (SVFs) isolation, using minimal and substantial manipulation methods, have been described [6‐11]. The European Medicines Agency (EMA), and the US Food and Drug Administration (FDA), described adult cells as biological products obtained from two different methods of manipulation: “Minimal Manipulation” and “Substantial Manipulation.” Minimal manipulation does not modify the biological characteristics and functions of cells. It is based on filtration, centrifugation, cutting decantation, fragmentation, cryopreservation, grinding, vitrification, shaping, freezing, lyophilization, soaking in antibiotics, cell separation/concentration/purification, irradiation, sterilization, without any cell expansion [6‐12]. Substantial manipulation is based on procedures of cell expansions and related cultures, cell differentiation, cell activation, genetic modification, and any processing that alters the biological, physiological, or structural characteristics of cells or tissues [11]. Minimal and substantial manipulation procedures have been detailed and described in the scientific literature, sometimes with conflicting results, presenting many different techniques that tried to find the best method of isolating SVFs and fat purification [6‐12]. Minimal manipulation procedures, based on centrifugation, filtration, washing, and fragmentation of the fat tissue, appeared as the most frequent strategies adopted to purify fat graft. In the last years, SVFs isolation procedures and lipofilling purification methods have deeply shifted from enzymatic digestion to mechanical processing [6‐12]. Enzymatic digestion of a tissue to release cells is considered to be substantial manipulation, when the aim is to dissociate cell-cell contacts, and the released cells are administered into patients with or without subsequent manipulation [13]. If the number of certain cells (e.g., mesenchymal stem cells—MSCs—in fat grafts) is enriched by selection and the processing does not change the characteristics of the cells, this is not considered a substantial manipulation [14]. Of the mechanical procedures, the washing seems to be that with less clinical data in terms of correlation between the SVFs amount, ADSCs number, and the fat volume maintenance [6‐12].

This article aimed to report the preliminary clinical results, represented by fat volume maintenance and ADSCs yield and growth, obtained by filtration and washing procedures of fat tissue.

The use of SVF in which ADSCs are contained is rapidly expanding in regenerative plastic surgery. As reported, the main method of fat digestion aiming to obtain SVF isolation is enzymatic, while the main strategies to obtain purified lipofilling are mechanical, each of them with several advantages and disadvantages [12].

Enzymatic digestion uses enzymes such as collagenase, trypsin, or dispase to dissociate the fatty tissue and release the mesenchymal stem cells. However, they are time-consuming, as they need an average of 60–90 minutes to digest the tissue and wash the enzymes out. Furthermore, enzymatic methods are more expensive than mechanical methods [12]. However, a great advantage of the enzymatic methods is the number of SVFs and ADSCs they yield. In conclusion, it could be argued that enzymatic methods are time-consuming, expensive, and require specific manipulations and knowledge [12]. On the contrary, mechanical processing allows the obtaining of purified lipofilling with a good amount of ADSCs within the SVF, with good viability, time-saving, and lower costs [12]. At the same time, mechanical procedures are based on different strategies (filtration, washing, fragmentation, centrifugation, etc.), some of them requiring special equipment and others being simple and cheap [12, 20, 21]. Most of them include some basic steps: washing, filtration, and centrifugation to concentrate the SVF and ADSCs into the purified fat [12, 20, 21]. The procedure described in the present paper manages to purify the lipofilling maintaining a good SVFs amount with excellent viability through simple and widely used means exclusively based on two procedures: washing and filtration. This procedure appears cheap, fast, and reliable, compared to other mechanical methods. The time needed to obtain lipofilling is short (ranging between 5 and 45 minutes, depending on the amount of injecting fat necessary), thus making mechanical methods ideal for one-stage procedures. The procedures are completely safe since they use autologous tissue, and the stem cells are not processed by any chemical or enzymatic means. Regarding mechanical processing, a recent study, showing the differences between mechanical and enzymatic procedures, in terms of SVFs amount and fat volume maintenance, was conducted by Gentile et al [12]. In this study, 80 patients affected by face and breast soft tissue defects were treated using different lipofilling procedures. In detail, 20 patients were treated using lipofilling enhanced with SVFs obtained by enzymatic digestion, 20 patients with SVFs obtained by centrifugation with filtration, 20 patients with SVFs obtained by only filtration, and 20 patients were treated with lipofilling obtained by only centrifugation according to the Coleman technique. In patients treated with lipofilling enhanced with SVFs obtained by automatic enzymatic digestion, a 63% ± 6.2% maintenance of fat volume restoration after 12 months was observed compared with 52% ± 4.6% using centrifugation with filtration, 39% ± 4.4% using only centrifugation (Coleman), and 60% ± 5.0% using only filtration [12]. Cell yield analysis showed that filtration was the most efficient system between mechanical digestion procedures thanks to the highest amount of SVFs obtained with fewer cell structure damage, producing the most volume maintenance after 12 months [12]. In the same line, in the present work, ADSCs seem to have an increased growth ability for the SVF obtained with the washing and filtration procedure, if compared to the standard Coleman technique based on centrifugation of 3000RPM (average 1100 g). The superiority of washing and filtration over centrifugation could be due to the mechanical issues of the g force, as suggested by Kurita et al [22]. Kurita et al. [22] investigated the effects of centrifugation at 400, 700, 1200, 3000, or 4200g for 3 minutes on liposuction aspirates reporting two important findings:

Therefore, excessive centrifugation can destroy adipocytes and ADSCs, but appropriate centrifugation concentrates them, resulting in enhanced fat graft maintenance. Kurita et al. [22] recommend 1200g as an optimized centrifugal force for obtaining good short- and long-term results in fat transplant.

Regarding the mechanical procedures based on washing, and purification of fat tissue, two different studies performed by Schafer et al. [23] and Zhu et al. [24] in which Puregraft^® was used, displayed augmented tissue viability, and reduced quantity of red blood cells, free lipids, and contaminants when compared to other lipofilling. Additionally, a study performed by Dos-Anjos Vilaboa et al. [25] displayed the results obtained by fat tissue washed and purified via GID 700™ displaying significantly reduced amounts of lactate dehydrogenase, triglycerides, and hematocrit maintaining the adipose graft osmolarity. SVFs obtained by the filtration device LipiVage™ displayed endothelial and mesenchymal progenitor cells, maintaining their differentiation capacity when used as fibrin spray [26]. Ferguson et al. [27] additionally reported that LipiVage™ produced a higher number of adipocytes and sustained a higher level of intracellular enzyme (glycerol-3-phosphatase dehydrogenase (G3PDH)) activity within fat grafts. A study performed by Bianchi et al. [28] reported that cells obtained from Lipogems^® showed a significantly higher concentration of mature pericytes, ADSCs, exosomes, and a lower quantity of hematopoietic cells when compared to isolated cells with enzymatic digestion. Condé-Green et al. [29] conducted a prospective cross-sectional study in which fat obtained by liposuction of the lower abdomen was separated and processed by decantation, washing, and centrifugation. Cell count per high-powered field of intact nucleated adipocytes was significantly greater in decanted lipoaspirates, whereas centrifuged samples showed a greater majority of altered adipocytes [29]. MSC concentration was significantly higher in washed lipoaspirates compared to decanted and centrifuged samples [29].

A very recent systematic review published in the current year 2023 by Langridge et al [30] confirmed the superiority of washing and filtration procedures. 24 studies (2413 patients) on fat processing techniques including centrifugation, decantation, washing, filtration, and gauze rolling, as well as ADSCs enrichment methods using commercial devices were identified and analyzed in this systematic review [30]. Complications were infrequent; palpable cysts (0–20%), surgical-site infections (0–8%), and fat necrosis (0–58.4%) were the most reported [30]. No significant differences in long-term volume maintenance between techniques were found in fat grafting of the breast. In head and neck patients, greater volume maintenance was documented in ADSCs enrichment (64.8–95%) and commercial devices (41.2%) compared to centrifugation (31.8–76% [30]). Langridge et al [30] concluded graft processing through washing and filtration, including when incorporated into commercial devices, results in superior long-term outcomes compared to centrifugation and decantation methods. ADSCs enrichment methods and commercial devices seem to have superior long-term volume maintenance in facial fat grafting [30].

As reported in the current work, and as analyzed from the above-mentioned studies a lack of a single standardized protocol both for the lipofilling purification and its enhancement with SVFs, must be considered as a limitation, as prevents having detailed predictability of the outcomes in terms of fat volume maintenance.

Mechanical methods for purifying adipose are safe, cost-effective, and suitable for one-stage procedures, favoring comfort for patients. The method based on the washing and filtration procedure here described appeared effective, quick, safe, and can be easily reproduced, compared with centrifugation both in terms of ADSCs amount, viability, and fat volume maintenance. Additional perspective studies and controlled trials will be necessary to confirm the preliminary data here reported.