Cell-Enriched Lipotransfer (CELT) Improves Tissue Regeneration and Rejuvenation without Substantial Manipulation of the Adipose Tissue Graft

The good availability and the large content of adult stem cells in adipose tissue has made it one of the most interesting tissues in regenerative medicine. Although lipofilling is one of the most frequent procedures in plastic surgery, the method still struggles with high absorption rates and volume losses of up to 70%. Therefore, many efforts have been made to optimize liposuction and to process the harvested tissue in order to increase fat graft retention. Because of their immunomodulatory properties, their cytokine secretory activity, and their differentiation potential, enrichment with adipose tissue-derived stem cells was identified as a promising tool to promote transplant survival. Here, we review the important parameters for lipofilling optimization. Finally, we present a new method for the enrichment of lipoaspirate with adipose tissue-derived stem cells and discuss the parameters that contribute to fat graft survival.


Introduction
Adipose tissue is one of the most promising sources of tissues and cells for regenerative medicine. It can be harvested with minimally invasive procedures and contains various cell types beneficial for tissue regeneration and wound healing [1][2][3]. For example, adipose tissue contains the highest number of mesenchymal stem cells (MSCs) per volume [4][5][6][7]. The MSCs of fat tissue, adipose tissue-derived stem cells (ADSCs), have the potential to differentiate into various cell lineages including the adipogenic, chondrogenic, osteogenic, neural, myogenic, and endothelial lineages [1,[8][9][10][11][12][13]. Additionally, ADSCs have immunomodulatory properties and secrete growth factors crucial for cell proliferation and angiogenesis [7,[14][15][16][17][18][19][20][21]. Therefore, autologous fat grafting, or lipofilling, has become one of the most frequent procedures in plastic surgery for aesthetic and reconstructive purposes [22][23][24][25]. It has been optimized since the first reports about the transfer of autologous fat in the 19th century [26,27], the first autologous fat injection with a needle in the early 20th century [28,29], and the establishment of modern liposuction in the late 20th century [30][31][32][33][34][35]. Today, lipofilling with autologous fat is a safe and relatively simple procedure, with no risk of immunogenicity and low costs [36][37][38][39][40]. However, graft survival and take rate remain hard to predict, with volume losses of up to 70% [41][42][43][44]. This poses the main challenge for the clinical application of lipofilling and autologous fat transfer in regenerative medicine. Therefore, a lot of effort has been put into developing methods to inflammation factors, cellular debris, and regeneratively useless erythrocytes [23,35,75]. It has been shown that the removal of these contaminants is beneficial for fat graft survival [23,[75][76][77]. The simplest method to achieve separation is sedimentation. Here, the lipoaspirate is allowed to separate into phases under gravitational force. An aqueous phase, being the densest phase, settles at the bottom of a given container. A lipid phase, being the least dense phase, settles as the top layer. In between is the desired adipose tissue. Sedimentation alone is sufficient to increase the percentage of target cells via separation from unwanted contaminants and to support graft survival [78]. This phase separation can be accelerated and strengthened by applying centrifugal forces. Centrifugation led to a higher concentration of adipocytes and ADSCs compared to sedimentation but did not further increase graft viability [35]. This might be related to the cell-survival hypothesis for spherical lipografts, which is discussed in more detail below [39,72,79]. Briefly, the enrichment of cells beyond sedimentation might be useless without decreasing the size of the transplanted particles. An alternative to sedimentation and centrifugation is filtration. Both automated and manual techniques are applied commonly. Although filtration techniques resulted in both the satisfactory removal of contaminants and a higher concentration in cellular components, the graft take rate was not improved in most studies [80][81][82][83][84][85].
Although sedimentation, centrifugation, and filtration remove lipids, water, and other contaminants, providing a tissue with an increased number of viable cells in general, a different approach of tissue processing is needed for enrichment with stem cells particularly. Most of the effects that are beneficial for fat graft survival depend on secreted factors [86]. Neovascularization and cellular regenerative potential have been identified as major requirements for the graft take rate [17]. Because ADSCs, like all MSCs, secrete growth factors for neoangiogenesis and cell survival, a higher number of stem cells per lipograft volume is a promising approach for take rate improvement. The obvious choice to achieve a higher stem cell number is simply the addition of culture-expanded ADSCs, a procedure known as cell-assisted lipotransfer (CAL) [87][88][89][90]. This method was shown to be effective in supporting fat graft retention [91][92][93] but includes the isolation and cultivation of stem cells, and therefore, bears the risk of regulatory restrictions. The enzymatic digestion of tissue [94] is considered a substantial manipulation, rendering the obtained cells an advanced-therapy medicinal product (ATMP) in Europe (1394/2007) and the USA (21 CFR 1271.10) [95]. The utilization of xenogeneic digestion enzymes or cultivation with xenogeneic culture sera may further prevent clinical usage of these cells [96,97]. Enzyme-free isolation techniques, such as explant cultures and defined serum-free cell culture media, could evade the problems that come along with xenogeneic substances [94]. However, the isolation and in vitro cultivation of cells might still be considered an inadmissible substantial manipulation. Additionally, the utilization of isolated and cultured autologous cells would necessitate a second surgical intervention. However, washing, rinsing, centrifuging, and filtration aren't considered substantial manipulation. Tissue preparation obtained in this way is suitable for structural support, and is therefore considered minimally processed [39].
Mechanical processing of lipoaspirate constitutes an alternative for the further enrichment of stem cells [77]. Here, the tissue is exposed to mechanical shear stress or other physical forces, which is not substantial manipulation [91]. An example of mechanical shear stress applied to lipoaspirate is intersyringe processing [98]. For this, the lipoaspirate is shifted quickly between two syringes through a connector with a given, usually small, diameter. This leads to decreased particle size and the rupture of many mature adipocytes without affecting the small and robust ADSCs. Table 1 summarizes the different tissue-processing methods. The method of intersyringe processing using the specific device, technology, and protocol presented, and its implications, will be discussed in more detail in the next section. However, an alternative for particle-size reduction is the utilization of a strainer of defined pore size [99]. Table 1. Different tissue-processing methods and the impact on different parameters. Centrifugation and filtration are better for the removal of contaminants than sedimentation, but do not increase graft viability without particle-size reduction. Mechanical processing in combination with centrifugation combines contaminant removal with the best stem cell enrichment and graft viability enhancement without regulatory restrictions (-: no impact; *: impact; **: stronger impact; ***: strongest impact).

Cell-Enriched Lipotransfer
Cell-Enriched Lipotransfer (CELT) is a sequence of simple consecutive steps that result in a stem cell-enriched tissue. This processing method can be combined with any harvesting technique and has a broad range of applications. However, we will give a brief overview of the harvesting procedure commonly used by us. A 0.9% (w/v) solution of NaCl (a volume equal to the volume of fat tissue to be harvested) containing epinephrine (1:200,000) is infiltrated over approximately 15 min according to the S2K guidelines [24] using a 2.5 mm injection cannula. The lipoaspirate is harvested using a 3.8 mm cannula. We utilize a machine for automated negative pressure (Body-Jet ® , Human Med AG, Schwerin, Germany), which allows water-jet-assisted liposuction with a constant negative pressure of less than 0.5 mbar. After the lipoaspirate is allowed to sediment, it is centrifuged at 1600× g for 2 min with brakes enabled. Subsequently, the tumescent layer and the lipid layer are discarded. For intersyringe processing, a connector with a diameter of 1.2 mm is used and the lipoaspirate is forced manually, 10 times in total, quickly through the connector. Afterwards, the processed tissue is once more centrifuged at 1600× g for 2 min and separated from the aqueous phase and the lipid phase. The total processing time is under ten minutes.
With the first centrifugation, the infiltrated tumescence solution is primarily separated from the tissue preparation. Only a small lipid layer from the adipocytes disrupted during the liposuction resulting from this centrifugation. However, it is crucial to remove this large amount of aqueous phase prior to the intersyringe processing. With the second centrifugation, the released lipids from disrupted mature adipocytes are primarily separated from the tissue preparation. Only a small aqueous layer from the adipocytes' cytosol emerges from the second centrifugation ( Figure 1). The resulting tissue contains highly concentrated ADSCs and other cells from the stromal vascular fraction in a massively reduced volume, with a reduced number of mature adipocytes and a reduced size of fat lobules. Although no individual step of the procedure is a novelty in its own right, the combination in the specified order and with the specified parameters results in a graft optimized for graft retention, graft survival, and tissue regeneration. This tissue preparation can be used directly for the treatment of low-volume defects for which it is especially suitable because it can be applied through very fine cannulas with diameters of less than one millimeter. However, it can also be used as an addition to one-time centrifuged lipoaspirate if larger volumes are needed, albeit with lower concentration of ADSCs. one millimeter. However, it can also be used as an addition to one-time centrifuged lipoaspirate if larger volumes are needed, albeit with lower concentration of ADSCs. We were able to demonstrate that our mechanical lipograft processing causes no substantial manipulation of the cells. ADSC viability and cytokine secretion were not compromised by the method [100]. The tissue structure is preserved, with significantly smaller particles. Additionally, the differentiation potential of ADSCs in the processed tissue was unchanged. Histologically, we were able to demonstrate that the mature adipocytes from large adipocyte aggregations were especially disrupted by the shear-force processing. Mature adipocytes adjacent to the vascular system or large extracellular matrix depositions were unaffected by the mechanical stress [101]. This is in accordance with a recently published hypothesis regarding the lobule organization of adipose tissue and its impact on regeneration potential [102]. Clinically, we were able to demonstrate excellent results with CELT lipografts for facial rejuvenation and other applications [103,104].
It is believed that transplant survival rates depend on the viability of grafted cells, neoangiogenesis, and the overall regeneration potential of the grafted cells [17,87,[105][106][107][108][109]. Therefore, the higher concentration of stem cells in CELT tissue could explain the increased graft retention we observed in the clinic. However, many studies with centrifuged, and therefore, cell-enriched lipoaspirate failed to show an increased take rate in comparison to sedimented lipoaspirate [110]. Recently, a cell-survival hypothesis for spherical lipografts was proposed [39,72,79]. Therein, it was hypothesized that only a thin outer layer (100 μm-300 μm) of the fat lobule in close proximity to oxygen and a nutrient supply can survive. In the middle zone, ADSCs may survive and proliferate but the central zone is mostly necrotic. Therefore, further stem cell enrichment by centrifugation beyond the sedimentation-induced cell concentration might be useless as long as the particle size is too big to allow for diffusion to the central zone. The ADSCs are also much more We were able to demonstrate that our mechanical lipograft processing causes no substantial manipulation of the cells. ADSC viability and cytokine secretion were not compromised by the method [100]. The tissue structure is preserved, with significantly smaller particles. Additionally, the differentiation potential of ADSCs in the processed tissue was unchanged. Histologically, we were able to demonstrate that the mature adipocytes from large adipocyte aggregations were especially disrupted by the shear-force processing. Mature adipocytes adjacent to the vascular system or large extracellular matrix depositions were unaffected by the mechanical stress [101]. This is in accordance with a recently published hypothesis regarding the lobule organization of adipose tissue and its impact on regeneration potential [102]. Clinically, we were able to demonstrate excellent results with CELT lipografts for facial rejuvenation and other applications [103,104].
It is believed that transplant survival rates depend on the viability of grafted cells, neoangiogenesis, and the overall regeneration potential of the grafted cells [17,87,[105][106][107][108][109]. Therefore, the higher concentration of stem cells in CELT tissue could explain the increased graft retention we observed in the clinic. However, many studies with centrifuged, and therefore, cell-enriched lipoaspirate failed to show an increased take rate in comparison to sedimented lipoaspirate [110]. Recently, a cell-survival hypothesis for spherical lipografts was proposed [39,72,79]. Therein, it was hypothesized that only a thin outer layer (100-300 µm) of the fat lobule in close proximity to oxygen and a nutrient supply can survive. In the middle zone, ADSCs may survive and proliferate but the central zone is mostly necrotic. Therefore, further stem cell enrichment by centrifugation beyond the sedimentation-induced cell concentration might be useless as long as the particle size is too big to allow for diffusion to the central zone. The ADSCs are also much more resistant to external conditions such as oxygen deprivation and radiotherapy, as shown in our studies [111][112][113]. In this cell-survival hypothesis of spherical grafting, the maximum size for fat particles is 1.5 mm before the innermost part is too far from the surface to escape necrosis [39]. However, several studies indicate that, although the particle size should not exceed a threshold that prevents diffusional supply, those remaining within the Cells 2022, 11, 3159 6 of 11 tissue structure have numerous benefits for the cells' regenerative properties. In contrast to isolated single cells, the cells in microfragmented fat tissue show extended survival and long-lasting anti-inflammatory activity [114]. Additionally, the cell-matrix contacts and the cell-cell contacts with other cells of the stromal vascular fraction promote the proliferative and angiogenic effects of ADSCs [95,115,116]. Additionally, the physical properties of adipose tissue contribute to the therapy of inflammatory-joint-like conditions such as osteoarthritis [117].
It has been shown that cells from microfragmented fat show increased activity of cytokine and growth-factor secretion in comparison to enzymatically obtained cells [118]. This further contributes to the increased therapeutic potential of mechanically processed adipose tissue [95]. Interestingly, besides the increased secretory activity, cells obtained from microfragmentation show a higher pericyte content than the stromal vascular fraction obtained from enzymatic digestion [118]. Pericytes exist ubiquitously in vascularized tissues throughout the human body [119][120][121]. Additionally, it has long been proposed that ADSCs have a pericyte origin [122][123][124][125][126]. With a high pericyte content in mechanically processed adipose tissue, new developments in the discussion about its homologous use ("fat in fat") and its regulatory indications might emerge. Although the debate about the universal perivascular origin of MSCs remains unresolved with opposing positions [127], the higher pericyte content might be able to expand the area of application for autologous fat grafting in homologous use.

Conclusions
In past decades, many efforts have been made to improve the take rate and survival of lipografts. The CELT method combines contaminant removal, stem cell concentration, and particle-size reduction to improve fat graft retention in a fast, reliable, and cheap way and is in accordance with all regulatory requirements. The method is evaluated regarding both the cellular characterization and the clinical outcome.