Here, we present human adipose tissue enzyme-free micro-fragmentation using a closed system device. This new method allows the obtainment of sub-millimeter clusters of adipose tissue suitable for in vivo transplantation, in vitro culture, and further cell isolation and characterization.
In the past decade, adipose tissue transplants have been widely used in plastic surgery and orthopaedics to enhance tissue repletion and/or regeneration. Accordingly, techniques for harvesting and processing human adipose tissue have evolved in order to quickly and efficiently obtain large amounts of tissue. Among these, the closed system technology represents an innovative and easy-to-use system to harvest, process, and re-inject refined fat tissue in a short time and in the same intervention (intra-operatively). Adipose tissue is collected by liposuction, washed, emulsified, rinsed and minced mechanically into cell clusters of 0.3 to 0.8 mm. Autologous transplantation of mechanically fragmented adipose tissue has shown remarkable efficacy in different therapeutic indications such as aesthetic medicine and surgery, orthopedic and general surgery. Characterization of micro-fragmented adipose tissue revealed the presence of intact small vessels within the adipocyte clusters; hence, the perivascular niche is left unperturbed. These clusters are enriched in perivascular cells (i.e., mesenchymal stem cell (MSC) ancestors) and in vitro analysis showed an increased release of growth factors and cytokines involved in tissue repair and regeneration, compared to enzymatically derived MSCs. This suggests that the superior therapeutic potential of microfragmented adipose tissue is explained by a higher frequency of presumptive MSCs and enhanced secretory activity. Whether these added pericytes directly contribute to higher growth factor and cytokine production is not known. This clinically approved procedure allows the transplantation of presumptive MSCs without the need for expansion and/or enzymatic treatment, thus bypassing the requirements of GMP guidelines, and reducing the costs for cell-based therapies.
Adipose tissue, long used as a filler in reconstructive and cosmetic surgery, has recently become more popular in regenerative medicine once recognized as a source for mesenchymal stem cells (MSCs)1. Lipoaspirates dissociated enzymatically into single-cell suspensions yield an adipocyte-free stromal vascular fraction (SVF) that is used unaltered in the patient or, more commonly, is cultured for several weeks into MSCs2.
However, enzyme dissociation ruptures the tissue microenvironments, secluding neighboring regulatory cells from presumptive regenerative cells that become considerably modified by in vitro culture. To avoid such experimental artifacts and consequent functional alterations, attempts have been made to process adipose tissue for therapeutic use while maintaining its native configuration as intact as possible3,4. Notably, mechanical tissue disruption has started to replace enzymatic dissociation. To this end, the full immersion closed system micro-fragments lipoaspirates into sub-millimeter, blood- and oil-free tissue clusters (e.g., Lipogems) via a sequence of sieve filtration and steel marble induced disruption3. Autologous transplantation of micro-fragmented adipose tissue, using this closed system technology, has been successful in multiple indications, spanning cosmetics, orthopedics, proctology and gynaecology4,5,6,7,8,9,10,11,12,13.
Comparison between human micro-fragmented adipose tissue (MAT) obtained with the closed system device and isogenic SVF revealed that with respect to vascular/stromal cell distribution and secretory activity in culture, MAT contains more pericytes, which are presumptive MSCs14, and secretes higher amounts of growth factors and cytokines15.
The present article illustrates the enzyme-free micro-fragmentation of human subcutaneous adipose tissue using a closed system device, and the further processing of such micronized adipose tissue for in vitro culture, immunohistochemistry and FACS analysis, in order to identify the cell types present and the soluble factors secreted (Figure 1). The described method safely generates adipose derived sub-millimeter organoids containing viable adipose tissue cell populations in an intact niche, suitable for further applications and studies.
Ethical approval for the use of human tissues in this research was obtained from the South East Scotland Research Ethics Committee (reference: 16/SS/0103).
1. Subcutaneous Abdominal Adipose Tissue Collection
NOTE: All instruments used in the manual lipo-aspiration procedure are provided by the manufacturer of the micro-fragmentation device.
2. Micro-fragmentation of the lipoaspirate
NOTE: This protocol is meant for the research use only. Micro-fragmentation is performed with the help of a commercially available device (Table of Materials).
3. Fluorescence Immunohistochemistry
4. Digestion of the micro-fragmented adipose tissue and cell isolation
5. Cell labelling and sorting
6. Cell culture
Mechanical dissociation of manual lipoaspirates resulted in the production of micro-fragmented adipose tissue (MAT), consisting of an aggregate of adipocytes enveloping a microvascular network (Figure 3). Immunofluorescence analysis of gelatin-embedded and cryofixed MAT highlights this structure, showing the vascular network marked by the endothelial cell marker Ulex europaeus agglutinin 1 (UEA-1) receptor mainly consisting of small, capillary-like vessels (Figure 4). Notably pericytes expressing NG2 or PDGFRβ are normally distributed, ensheathing endothelial cells (Figure 4). These data confirm that the mechanical micro-fragmentation process is not affecting the perivascular cell compartment. The presence of perivascular cells, both pericytes and adventitial cells, has been confirmed by flow cytometry. To select cells a forward scatter area (FSC-A) vs side scatter area (SSC-A) gate was used (Figure 2A). The identified cell population was further gated using forward scatter area (FSC-A) vs forward scatter height (FSC-H) to select single cells and live cells were identified as negative for DAPI staining (Figure 2B-C). CD31 and CD45 markers were used to exclude hematopoietic and endothelial cells (Figure 2C). Finally, pericytes were identified as CD146+ CD34– and adventitial cells as CD146– CD34+ (Figure 2D). The gating strategy is summarized in Figure 2. The same gating strategy was followed for sorting experiments.FACS-purified MAT pericytes and adventitial cells both exhibit a spindle to stellate morphology in culture. Culture for 8 days showed that MAT secretes more cytokines (adiponectin, CD14, CD31, CD154, chitinase 3-like 1, complement factor D, CD147, GDF-15, IGFBP-2, IL1RA, IP-10, M-CSF, MIF, CXCL9, CCL19, PDGFAA, CCL5, RBP-4, relaxin-2, ST2, TNF-α, CD87), and angiogenic factors (angiogenin, angiostatin, DPPIV, endoglin, HGF, leptin, PDGFAB/BB, PlGF, thrombospondin 2, TIMP4, uPA) in vitro than SVF. Moreover, cytokines and angiogenic factors produced by both MAT and SVF were more abundant in MAT supernatants. Accordingly, MAT digested with collagenase and placed in culture produced a secretome similar to that observed from conventional SVF15. Globally, all these data demonstrate that micro-fragmented adipose tissue is not only therapeutically advantageous, but also amenable to phenotypic and functional investigations. In particular, the small size of MAT clusters allows testing of secretory activity in culture, which would be more difficult with large adipose tissue chunks.
Figure 1: Schematic representation of the micro-fragmentation of subcutaneous adipose tissue. Lipoaspirate is collected from subcutaneous adipose tissue using a cannula. Collected lipoaspirate is micro-fragmented using the close system device. Resulting micro-fragmented adipose tissue (MAT) is composed by multiple cell types including perivascular cells and adipocytes. MAT can be used in multiple tests, including explant culture and immunohistochemistry, and for cell isolation. Please click here to view a larger version of this figure.
Figure 2: Representative gating strategy for perivascular cell analysis/sorting from MAT. A forward scatter area (FSC-A) vs side scatter area (SSC-A) gate is used to identify cells (A), followed by forward scatter area (FSC-A) vs forward scatter height (FSC-H) to select single cells (B). Viable non-endothelial and non-hematopoietic cells are gated as negative for DAPI, CD31-V450 and CD45-V450 respectively, in the same panel (C). Finally, perivascular cells are identified as pericytes (CD146+ CD34–) or adventitial cells (CD146– CD34+) (D). Please click here to view a larger version of this figure.
Figure 3: MAT morphology. Bright-field view of MAT cultured in basal medium. Scale bar = 1,000 µm. Please click here to view a larger version of this figure.
Figure 4: Vasculature network in MAT. Endothelial cells are stained with UEA-1. Boxed areas in A are showed enlarged in B and C. Arrowheads indicate pericytes, which have been stained using antibodies against PDGFRβ and NG2. Scale bar = 50 µm. Please click here to view a larger version of this figure.
This paper describes the physical fractionation, using a closed system device, of human adipose tissue into small clusters displaying normal adipose tissue microanatomy.
Manually aspirated human subcutaneous adipose tissue and saline solution are loaded into a transparent plastic cylinder containing large pinball-style metallic spheres that, upon vigorous manual shaking of the device, rupture the fat into sub-millimeter fragments. Attached filters and outlet allow eliminating debris, blood and free lipids and MAT is collected in a syringe linked to the device. Here, MAT has been successfully further processed for immunohistochemistry, flow cytometry and culture, although this specific device was developed in a medical perspective, to replace in the theatre the enzymatically produced adipose tissue stromal vascular fraction or culture derived adipose stem cells, and indeed proved highly efficient in plastic surgery and diverse other cell-based therapies4,5,6,7,8,9,10,11,12,13.
Therefore, adipose tissue long used intact as a mere filler16 returns in its native 3-dimensional configuration, after years of enzymatic dissociation and in vitro culture. This follows a general trend to grow and study tissues in their innate multicellular arrangement rather than as dispersed cell populations, as illustrated by the growing popularity of organoids17.
Adipose tissue fragmentation using this mechanical device has proven reliable and reproducible and no modification of the original protocol was ever necessary. The ordinary supply of human subcutaneous fat for experimental studies is a lipoaspirate collected for cosmetic reasons, which is loaded directly onto the fragmentation device. A limitation for users in the research laboratory, though, is that the adipose tissue to be processed must be gently aspirated by hand, to maintain tissue microarchitecture as intact as possible, and not suck out with a vacuum pump as is routinely done in the operating room. Unless a surgeon willing to collect fat manually can be identified, a freshly resected abdominoplasty residue can be used. The fat attached to the inner aspect of the abdominal skin can then be carefully and sterilely aspirated with the syringe provided with the kit. This is the reason why we have described this step at the beginning of the protocol (step 1).
What actually explains the therapeutic superiority of MAT over enzyme dissociated adipose tissue? Flow cytometry analysis of MAT enzymatically dissociated immediately after fractionation revealed a higher proportion of pericytes, known as innate forerunners of mesenchymal stem cells14, as compared with total aspirated adipose tissue15. More directly, the comparative analysis of MAT and SVF culture supernatants showed that the former secretes, qualitatively and quantitatively, more growth factors and cytokines involved indirectly in tissue repair. These include pro-angiogenic factors, as well as diverse mediators of immuno-inflammation15.
Beyond these recent results, much remains to be understood regarding the molecular basis of MAT therapeutic potential. To this end, the method here described represents a simple, fast technique that is low-impact to the adipose niche and generates MAT ideal for further experimental manipulation. From an ancillary point of view, research is also conducted to improve the usability of MAT in the clinic; in this respect, determining whether micro-fragmented adipose tissue can be frozen for delayed autologous or allogeneic administration appears as a priority. Finally, it will be important to determine whether other tissues and organs such as the bone marrow, pancreas or skeletal muscle, to cite but a few, can be micro-fragmented with the same device and provide intact microvasculature associated cellular niches amenable to experimental intervention.
The authors have nothing to disclose.
The authors wish to thank Claire Cryer and Fiona Rossi at the University of Edinburgh for their expert assistance with flow cytometry. We also wish to thank the personnel of the Murrayfield hospital who contributed by providing tissue specimens.
This work was supported by grants from the British Heart Foundation and Lipogems, which supplied adipose tissue processing kits. Human adult tissue samples were procured with full ethics permission of the South East Scotland Research Ethics Committee (reference: 16/SS/0103).
4% Buffered paraformaldehyde (PFA) | VWR chemicals | 9317.901 | |
0.9% NaCl Solution | Baxter | 3KB7127 | |
AlexaFluor 555 goat anti-mouse IgG | Life Technologies | A21422 | |
AlexaFluor 647 goat anti-Rabbit IgG | Life Technologies | A21245 | |
Ammonium chloride | fisher chemicals | 1158868 | |
Antigent Diluent | Life Technologies | 3218 | |
Anti-Mouse Ig, κ/Negative Control (BSA) Compensation Plus | BD Biosciences | 560497 | |
Avidin/Biotin Blocking Kit | Life Technologies | 4303 | |
BD LSR Fortessa 5-laser flow cytometer | BD Biosciences | Laser 405nm (violet)/375nm (UV) – filter V450/50 for DAPI and V450 antibodies; Laser 561nm (Yellow-green) – filter YG582/15 for PE antibodies; Laser 405nm (violet)/375nm (UV) – filter V710/50 for BV711 antibodies | |
Biotinylated Ulex europaeus lectin | Vector Laboratories | Vector-B1065 | |
BV711 Mouse IgG1, k Isotype Control | BD Biosciences | 563044 | |
CD146-BV711 | BD Biosciences | 563186 | |
CD31-V450 | BD Biosciences | 561653 | |
CD34-PE | BD Biosciences | 555822 | |
CD45-V450 | BD Biosciences | 560367 | |
DAPI | Life Technologies | D1306 | stock concentration: 5mg/mL |
Disposable liposuction cannula (LGI 13Gx185 mm – AR 13/18) | Lipogems | provided in the Lipogems surgical kit | |
Diva software 306 (v.6.0) | BD Biosciences | ||
DMEM, high glucose, GlutaMAX without sodium pyruvate | Life Technologies | 61965026 | |
EGMTM-2 Endothelial Cell Growth Medium-2 BulletKitTM | Lonza | CC-3156 | |
Fetal Calf Serum (FCS) | Sigma-Aldrich | F2442 | |
FlowJo (v.10.0) | FlowJo | ||
Fluoromount G | SouthernBiotech | 0100-01 | |
Gelatin | Acros Organics | 410870025 | |
Lipogems Surgical Kit | Lipogems | LG SK 60 | |
Mouse anti human- NG2 | BD Biosciences | 554275 | stock concentration: 0.5 mg/mL |
PE Mouse IgG1, κ Isotype Control | BD Biosciences | 555749 | |
Penicillin-Streptomycin | Sigma-Aldrich | P4333 | |
Phosphate buffered saline (PBS) | Sigma-Aldrich | D8537 | |
Polystirene round bottom 5 mL tube with cell strainer snap cap | BD Biosciences | 352235, 25/Pack | |
Polystyrene round bottom 5 mL tubes | BD Biosciences | 352003 | |
Rabbit anti human – PDGFRb | Abcam | 32570 | stock concentration: 0.15 mg/mL |
Streptavidin conjugated-488 | Life Technologies | S32354 | |
Sucrose | Sigma-Aldrich | 84100-5kg | |
Tissue infiltration cannula (17GX185 mm-VG 17/18) | Lipogems | provided in the Lipogems surgical kit | |
Tris base | fisher chemicals | BP152-500 | |
Type- II Collagenase | Gibco | 17101-015 | |
V450 Mouse IgG1, κ Isotype Control | BD Biosciences | 560373 | |
Widefield Zeiss observer | Zeiss | Objective used: Plan-Apo 20x/0.8 | |
Zeiss Colibri7 LED light source ( LEDs: 385, 475, 555, 590, 630 nm) | Zeiss | DAPI: UV, excitation 385nm; 488: Blue, excitation 475nm; 555: Green, excitation 555nm; 647:Red, excitation 630nm |