Innovative Adipose Tissue Fractionation for Transforming Fat into Specialized Components

Summary

Here, we present a protocol that can transform autologous adipose tissue into various adipose component substances with different physicochemical properties and functions.

Abstract

Traditional autologous adipose tissue grafting is commonly used for volume augmentation and tissue regeneration. However, the uniform physical and biological properties of fat grafts often lead to suboptimal performance in diverse patient scenarios and multifaceted requirements. Expanding upon the foundations of autologous fat transplantation, we introduce a pioneering approach termed autologous adipose component transplantation. This innovative concept integrates a meticulously designed protocol aimed at converting autologous adipose tissue into a diverse array of adipose components, each characterized by unique physicochemical properties tailored to address specific applications across varied clinical contexts. Through a series of physical methods, adipose tissue can be transformed into an Adipose Matrix Complex (AMC) providing sufficient rigid support, High-Density Fat (HDF) for further filling, Stromal Vascular Fraction (SVF)-gel for small and/or precise injections, and Adipose Collagen Fragment (ACF) for fine wrinkles and/or facial rejuvenation needs. These distinct adipose components possess inherent characteristics that lend themselves to precise and targeted transplantation strategies, catering to the nuanced demands of individual patient needs. The novel adipose tissue fractionation protocol shows great potential in revolutionizing adipose tissue grafting. It offers a new perspective and methodology for more refined and tailored transplantation.

Introduction

Autologous adipose tissue grafting is a widely used technique for volumetric enhancement and tissue regeneration, driven by advancements in donor site preparation, fat extraction, processing, and implantation^1,2,3,4. However, the uniform physical and biological properties of traditional fat grafts often limit their effectiveness in addressing diverse clinical needs⁵. To overcome these limitations, our research team has developed an innovative adipose tissue fractionation protocol, transforming autologous fat into specialized components with distinct physicochemical properties and functions.

The primary goal of the protocol is to fractionate autologous adipose tissue into specialized components -- Adipose Matrix Complex (AMC), Stromal Vascular Fraction (SVF)-gel, and Adipose Collagen Fragment (ACF), each designed to address specific clinical challenges. By providing components with unique properties, such as enhanced rigidity, high cell density, and collagen-rich formulations, the protocol enables precise and targeted transplantation strategies^{6,7,8,9,10,11}.

Traditional fat grafting, while effective for volume augmentation, often fails to meet the nuanced demands of complex clinical scenarios. Its unpredictable fat absorption leads to unstable volume restoration. Moreover, due to the uniform physical and biological properties of traditional fat grafts, they cannot provide targeted filling for areas with specific transplantation requirements, such as structural support, performing precise small-area injections, and achieving targeted collagen stimulation. These issues frequently necessitate multiple surgeries, thus increasing patients' pain and financial burden^1,2,5. This fractionation protocol addresses these limitations by specifically enriching adipose tissue with content of particular value from traditional adipose tissue. Through this process, we form adipose tissue components with tailored properties, thus offering clinicians a versatile toolkit for personalized treatment. Compared to synthetic fillers or traditional fat grafting, the approach utilizes autologous tissue, eliminating the risk of immune rejection and ensuring biocompatibility³. Additionally, the fractionation process creates components with unique properties, such as AMC, which provides structural support and features a low absorption rate; SVF-gel, which provides precise injections and is characterized by a high density of stromal vascular fraction cells; and ACF, a collagen protein scaffold capable of controlled release of fat factors, which shows potential for clinical applications in skin rejuvenation^7,9,11. This approach also reduces the need for repeated procedures by improving graft retention and patient satisfaction^10,12.

The concept of fractionating biological tissues to optimize therapeutic outcomes is well-established, as seen in the separation of blood into plasma, red blood cells, and platelets for targeted medical applications. In recent years, research and clinical applications of adipose tissue components have also been gradually advancing^{13,14,15,16,17,18}. This adipose fractionation protocol builds on this principle, isolating components with distinct properties for tailored clinical use. Clinicians can determine the suitability of this method by considering the patient's anatomical needs, desired outcomes (e.g., volume augmentation, structural support, or skin rejuvenation), and available equipment. This protocol is particularly effective for cases requiring precise control over graft properties, such as facial rejuvenation, scar revision, or complex reconstructive procedures^7,10,12.

Protocol

This research was approved by the Ethical Review Board in Nanfang Hospital, Guangzhou, China. Adipose tissue was collected from healthy donors who gave written informed consent to take part in the study. All animal experiments were approved by the Nanfang Hospital Institutional Animal Care and Use Committee and performed according to the guidelines of the National Health and Medical Research Council (China).

1. Fat preparation

1. General anesthesia and tumescent fluid infiltration
   1. Place the patient under general anesthesia. During the intravenous induction phase, administer a combination of propofol (2 mg/kg), fentanyl (1.5 µg/kg), and rocuronium (0.6 mg/kg), followed by maintenance of anesthesia depth using sevoflurane (1.5%) and remifentanil (0.1 µg/kg/min). Throughout the procedure, dynamically adjust the anesthesia plan based on BIS monitoring (target value 40-60), circulatory parameters (MAP >65 mmHg), and EtCO₂ (35-45 mmHg).
   2. Place the patient in the supine position with protective padding at pressure points and disinfect the surgical site rigorously with chlorhexidine-alcohol. Adhere to aseptic protocols to minimize the risk of infection.
   3. Infiltrate the subcutaneous tissue of the liposuction area (such as the abdomen or thigh) with tumescent fluid containing 0.06% lidocaine and 0.001% epinephrine using a 20 mL syringe. Maintain a 2:1 ratio of infiltrate to the anticipated volume of fat extraction, adjusting the total injection volume according to the patient's specific anatomical characteristics and the size of the treatment area.
   4. Check the anesthetic effect to confirm full tumescent fluid infiltration. Before the next step, prick or pinch the liposuction area. If the patient feels only slight pressure, infiltration is sufficient, enabling fat collection with less discomfort.
2. Fat harvesting
   1. Use a 3 mm multiport cannula with sharp side holes of 1 mm in diameter for fat collection. Perform the operation at −0.75 atm of suction pressure to minimize damage to the fat tissue and ensure the integrity and viability of the collected fat cells.
3. Coleman fat preparation
   1. Allow the collected fat, which is placed in a 60 mL syringe, to stand still for 10 min to let the liquid components settle to the bottom of the container.
   2. Carefully discard the liquid portion at the bottom and collect the upper, relatively pure fat layer to remove impurities such as blood and tumescent fluid.
   3. Retain the collected fat in the 60-milliliter syringe and centrifuge at 1,200 x g for 3 min at room temperature to further stratify the components.
   4. After centrifugation, discard the liquid portion at the bottom again. Retain the upper layer of fat, which is defined as Coleman fat, in the 60-
      milliliter syringe.
4. High- and Low -density fat sample collection
   1. Transfer the upper 2/3 of the Coleman fat into a 20 mL test tube, defining it as low-density fat (LDF).
   2. Transfer the remaining lower 1/3 of the Coleman fat into another 20 mL test tube, defining it as high-density fat (HDF).

2. Component extraction

1. Adipose matrix complex (AMC) preparation
   1. Pass the HDF through a filtering device with a sleeve and three internal sieves (1.5 mm filter screen spacing).
   2. Collect the attached tissue and dehydrate it in a 100-mesh filter bag surrounded by gauze. Determine the endpoint by observing the tissue as it is picked up with forceps; when no obvious liquid droplets fall upon forceps pickup, it indicates that the dehydration has achieved the desired effect. The final product was identified as AMC. Store the remaining HDF for future use.
2. Stromal vascular fraction (SVF)-gel extraction
   1. Mechanical emulsification of LDF: Mechanically emulsify the LDF by passing it between two syringes connected by a female-to-female Luer-Lok connector with an internal diameter of 1.4 mm at a rate of 10 mL/s for 1 min.
   2. Centrifuge the emulsified LDF at 1,600 x g for 3 min. Collect the middle layer, which resembles a gel-like substance, as SVF-gel.
3. Adipose collagen fragment (ACF) production
   1. Pulverization of LDF: Pulverize the LDF in a specialized ACF extractor at 30,000 rpm for 60 s to obtain a fat suspension.
   2. Filtering: Transfer the fat suspension into 20 mL syringes. Connect it to a 0.25 mm unidirectional filter, push the piston, and collect the filtrate in another 20 mL syringe. Then, connect this syringe to a 0.15 mm unidirectional filter, push the piston again, and collect the final filtrate in a third 20 mL syringe. Then, centrifuge the fat suspension at 3,000 x g for 3 min.
   3. Collect the solid portion at the bottom as ACF.

3. Testing of fat components

1. Stiffness testing
   1. Use a device with a force measuring sensor having a range of 0 N to 225 N and an electromagnetic actuator allowing for 6 mm of axial travel.
   2. Clamp each tissue sample (AMC and Coleman fat) and apply a preload of 25 - 30 N to eliminate slack and cause elongation. Set ramp loading at 8 mm/s and record the highest force achieved before the sample ruptured electronically. The device then calculates tissue stiffness.
2. Hematoxylin and eosin (HE) and Masson staining
   1. Fix the samples in 4% paraformaldehyde to preserve their structure. Dehydrate in ethanol gradient to remove water, which is necessary for the subsequent embedding process.
   2. Embed the samples in paraffin, a process that makes the samples easier to cut into thin sections. Next, section the paraffin-embedded samples.
   3. Stain the sections with HE and Masson's trichrome stains. Examine the samples using Scanning electron microscopy (SEM).
3. Transplantation studies
   1. Select 70 nude mice of Health SPF level, weighing 15 - 18 g and aged 4 - 6 weeks (irrespective of sex). Keep the mice in accordance with institutional guidelines, housed in individual cages with a 12 h light/dark cycle, and provided with standard food and water ad libitum.
   2. Anesthetize mice by intraperitoneal injection of pentobarbital sodium (50 mg/kg). Check the depth of anesthesia using the toe pinch reflex. Apply eye ointment to prevent dryness.
   3. Inject 0.3 mL of prepared AMC or adipose tissue (Coleman fat in the control group) into the subcutaneous tissues of each mouse, serving as the fat graft baseline. Monitor the animals until they regain consciousness and then transfer them to their cages.
   4. On Days 7, 14, 30, 60, and 90 post-grafting, harvest the grafts by carefully separating them from the surrounding tissue. Measure their volumes. The retention rate of grafts was defined as follows:
      retention rate (%) = the graft volume at harvest time/injection fat volume (0.3 mL here) x 100%.

Results

Through various mechanical procedures performed during the surgical process, we successfully transformed adipose tissue into distinct adipose components with different characteristics. They are, in sequence, the AMC that provides ample robust support (Figure 1A), the HDF for further filling (Figure 1B), the SVF-gel for precise injections in small areas (Figure 1C), and the ACF for addressing fine wrinkles or facial restoration needs...

Discussion

This adipose tissue fractionation protocol involves several critical steps that ensure the successful isolation of specialized components. First, the centrifugation of harvested adipose tissue separates it into high-density fat (HDF) and low-density fat (LDF), each with distinct cellular and extracellular matrix (ECM) compositions^23,24. HDF, rich in viable adipocytes and stromal vascular fraction (SVF) cells, is further processed to obtain the Adipose Matrix Comp...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.001% Epinephrine	Sigma-Aldrich	E4375
0.06% Lidocaine	Sigma-Aldrich	L5647
3-mm Multiport Cannula	Tulip Medical Products, San Diego, CA, USA	/
50-ml Centrifuge Tubes	Corning	430829
BOSE ElectroForce Load Testing Device	Bose Corporation	/
Collagenase	Worthington Biochemical	LS004176
Female-to-Female Luer-Lok Connector	BD Biosciences	309628
Filtering Device with Sleeve and Sieves	Custom-made (Shanghai Tiangong Instruments Co., Ltd.)	/
Gauze	Medline Industries	MDS093944
Hematoxylin/Eosin Staining Kit	Sigma-Aldrich	HT110116
Masson's Trichrome Staining Kit	Sigma-Aldrich	HT15
Nude mice	Southern Mdical University	/
Phosphate-Buffered Saline (PBS)	Thermo Fisher Scientific	10010023
Round Stainless-Steel Filter Screen (0.15 mm)	Shanghai Tiangong Instruments Co., Ltd.	/
Round Stainless-Steel Filter Screen (0.25 mm)	Shanghai Tiangong Instruments Co., Ltd.	/
Sterilized 100 Mesh Filter Bag	Shanghai Tiangong Instruments Co., Ltd.	/
Stromal Vascular Fraction (SVF)-Gel Extractor	Shanghai Tiangong Instruments Co., Ltd.	/
Trypsin	Thermo Fisher Scientific	25200056
Western Blot Analysis Kit	Bio-Rad	1705061