Efficient Isolation of Adipose-derived Stem Cells and Adipocytes from Porcine Adipose Tissue

Summary

Here, we present a standard method for efficiently isolating adipose-derived stem cells and adipocytes, characterized by >90% reduction in processing time, high cell viability, and broad compatibility.

Abstract

Adipose-derived stem cells (ADSCs) have emerged as ideal seed cells in regenerative medicine due to their abundant sources, minimally invasive harvesting, multi-lineage differentiation potential, and immunomodulatory properties. Their applications span tissue repair, disease modelling, and cell therapy; however, efficient isolation of high-viability ADSCs remains critical for advancing research and clinical translation. Conventional isolation methods, such as enzymatic digestion combined with mechanical dissociation via pipetting, are limited by lengthy processing times (1–3 h), poor cell viability (often <70%), and substantial batch-to-batch variability, compromising downstream experiments.

Here, we present the SoniConvert system (a mechanical wave-based cell separation system), which combines mechanical wave and enzymatic digestion to address these challenges. The system integrates a microprocessor-based control unit that regulates mechanical wave-mediated dissociation via digital feedback, together with a tissue-specific loosening reagent optimized for adipose tissue (5–15 min incubation). This approach enables rapid conversion of tissue to a single-cell suspension, with mechanical dissociation completed in 3–9 s.

The system offers three advantages: (1) ultra-fast processing, with the core isolation process—from enzymatic digestion to initial cell fraction separation—completed in approximately 30 min (>90% reduction compared to conventional methods); (2) high viability preservation, with trypan blue staining confirming cell viability of 80–95%, exceeding traditional protocols; and (3) broad compatibility, as the resulting single-cell suspensions meet requirements for primary cell culture, flow cytometry, cytotoxicity assays, and adipose organoid construction. By reducing processing time, enhancing cell integrity, and limiting variability, this system provides a practical platform for adipose-related research.

Introduction

Adipose-derived stem cells (ADSCs), a critical subset of mesenchymal stem cells (MSCs) isolated from adipose tissue, exhibit substantial therapeutic potential due to their accessibility, minimally invasive harvesting, robust proliferative capacity, multi-lineage differentiation potential, and potent paracrine and immunomodulatory properties^1,2,3,4. These attributes have positioned ADSCs as promising candidates for regenerative medicine, tissue engineering, and immunotherapy. However, the translational utility of ADSCs hinges on the ability t....

Protocol

All animal research was conducted in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals (Ministry of Science and Technology, China, revised in March 2017), and approved by the Animal Ethical and Welfare Committee of Sichuan Agricultural University (Permission number: 20230089). This protocol requires the use of the SoniConvert system, a commercially available device for mechanical wave‑assisted tissue dissociation (hereafter referred to as the mechanical dissociation system). The protocol is optimized for porcine adipose tissue, and its reliance on this specialized equipment is a key limitation.

Results

Figure 1: Schematic illustration of the adipose-derived cell isolation process. The diagram depicts the traditional method and the present method as illustrative workflows. Three main steps are involv.......

Discussion

The present study introduces a novel integrated approach for isolating adipose-derived cells (mainly adipocytes and ADSCs) that addresses critical limitations of conventional methods. By combining tissue-specific enzymatic loosening with precision mechanical wave dissociation, this system achieves rapid (total processing time ~30 min), high-viability (80–95%), and reproducible cell isolation.

Conventional enzymatic digestion, while widely used, relies on prolonged collagenase incubation .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
A Type Tissue Dissociation Kit	Docsense	TM001	Contains A Type Tissue Dissociation Agent (A-Type Solution) and A Type Tissue Dissociation Buffer (A-Buffer).
Adipogenic Differentiation Medium Kit	OriCell	GUXMD-90031	Used for adipogenic differentiation, including high-glucose Dulbecco's modified Eagle's medium (unmodified) and maintenance medium
Alizarin Red S	OriCell	A5533ALIR-10001	Mineralization staining for osteogenic differentiation(pH 5.2)
Ascorbic acid	Sigma-Aldrich	A4544	L-Ascorbic acid, cell culture tested, for osteogenic induction
Bama minipigs	Chengdu Dossy Experimental Animals Co.,Ltd		One year old, male, SPF-grade animals maintained under standardized vaccination, deworming, and cleaning procedures.https://www.cd-dossy.cn/animalpl_detail/12606067609851863 04.html
Bio-Rad TC20 Automated Cell Counter	Bio-Rad Laboratories	1450011	Automated cell counter for viability and yield assessment
BODIPY	Thermo Fisher Scientific	D3922	Neutral lipid stain
BSA (5%)	Sigma-Aldrich	A9418	Bovine serum albumin, lyophilized powder, for blocking
Calcitriol	Sigma-Aldrich	C9756	1α,25-Dihydroxyvitamin D3, for osteogenic induction
cell sieve (70 μm)	Corning	352350	Falcon 70 μm cell strainer, sterile, for FACS sample prep
cell strainer (200 μm)	pluriSelect	43-50200-03	pluriStrainer, PET mesh, fits 50 mL tubes, sterile
centrifuge tube (50 mL)	Corning	352098	Falcon 50 mL high clarity conical tube, sterile, polypropylene
Counting slide (for Bio-Rad TC20)	Bio-Rad Laboratories	1450011	Dual-chamber counting slide for TC20 automated cell counter
culture dish (100 mm)	Corning	430167	Sterile polystyrene dish, for hanging drop organoid formation
Dexamethasone	Sigma-Aldrich	D4902	≥98%, for adipogenic and osteogenic induction
D-Hanks Balanced Salt Solution or PBS	Gibco	10010015	Pre-cooled to 4 °C, pH 7.4.
DMEM	Gibco	11965084	This is used for fresh isolated SVF cultivation
DNase I	Beyotime	D7073	Optional, recommended if clumping is observed.
ethanol	Sigma-Aldrich	459836	75% for surface disinfection.
Fetal Bovine Serum (FBS)	Gibco	A4736201	Tet system approved, US origin, sterile
Flow Cytometer	BD Biosciences	BD FACSCanto II	3 lasers (405 nm, 488 nm, 633 nm), 8 fluorescence channels, for surface marker analysis (CD73, CD90, CD11b, CD45), meets 70 m nozzle requirement at 20 psi
fluorescence microscope	Olympus		Inverted or upright model capable of FITC and DAPI filter sets.
Fluorescent secondary antibodies	Thermo Fisher Scientific	A-11001 (anti-mouse), A-11008 (anti-rabbit)	Alexa Fluor conjugated, for immunofluorescence
Fluorochrome-conjugated antibodies (CD73, CD90, CD11b, CD45)	BD Biosciences	561254 (CD73), 561970 (CD90), 557321 (CD11b), 555485 (CD45)	For flow cytometric analysis of ADSC phenotype
Hoechst	Thermo Fisher Scientific	H3570	Nuclear stain for live/dead cell assessment.
IBMX	Sigma-Aldrich	I5879	≥99% (HPLC), for adipogenic induction medium
Ice-cold sterile saline	Baxter	2F7124	0.9% Sodium Chloride Injection, USP, for rinsing tissue
Insulin	Sigma-Aldrich	I9278	Recombinant, expressed in yeast, for adipogenic induction
iodophor	Sigma-Aldrich	25104	2% disinfectant solution.
Matrigel	Corning	354230	Growth factor reduced, phenol red-free, for embedding spheroids
Metal Bath	Servicebio	SMB-H	Used for incubation at 37 °C.
Oil Red O	OriCell	OILR-10001	Lipid staining for adipocyte differentiation
optical microscope	Olympus		Standard brightfield microscope with 10x–40x objectives.
Osteogenic Differentiation Medium Kit	OriCell	GUXMD-90021	Used for osteogenic differentiation, including high-glucose Dulbecco's modified Eagle's medium (unmodified) and maintenance medium
P1000 pipette with wide-bore tips	Beckman Coulter	B01113	Biomek P1000 Span-8 wide bore tips, sterile
Paraformaldehyde (4%)	Thermo Fisher Scientific	J61899	Aqueous solution, for fixing organoids
pasteur pipette	Corning	7095S-5C	Sterile, disposable.
Penicillin-Streptomycin	Gibco	15070063	10,000 U/mL penicillin, 10,000 μg/mL streptomycin, sterile
Pentobarbital sodium	Hikma Pharmaceuticals USA Inc.	24201-010-20	Injectable, for euthanasia (100 mg/kg body weight, intravenous). ANDA 203619
Primary antibodies (CD73, CD90, CD11b, CD45)	BD Biosciences	561254 (CD73), 561970 (CD90), 557321 (CD11b), 555485 (CD45)	For organoid immunostaining
Red Blood Cell (RBC) Lysis Buffer	Solarbio	R1010	If required for downstream applications.
Refrigerated centrifuge	Eppendorf	5702R	Used for centrifugation steps at 4 °C.
Rosiglitazone	Cayman Chemical	71740	≥98%, PPARγ agonist for adipogenic induction
Sharps container	Becton Dickinson	305270	Puncture-resistant container for used blades and scissors
SoniConvert Single Cell Suspension Preparation System	Docsense	SC-L1W	Used for mechanical dissociation of tissue.
Staining Buffer (Flow Cytometry Staining Buffer)	eBioscience (Thermo Fisher Scientific)	00-4222-57	200 mL, ready-to-use, contains FBS and 0.09% sodium azide as preservative, for antibody and cell dilution, surface staining and washing steps in flow cytometry
Sterile 1.5 mL Eppendorf tube	Eppendorf	30121.517	Safe-Lock microcentrifuge tube, sterile, for tissue mincing
Sterile culture dish	Corning	430167	100 mm x 20 mm sterile culture dish, for tissue collection
Sterile forceps	Sklar	96-1719	Sterile thumb forceps, serrated, for tissue handling
Sterile gauze	Dukal Corporation	7060033	Sterile gauze pads, for disinfection and wiping
Sterile ophthalmic scissors	Sklar	10023-580	Vannas-style spring scissors, for mincing tissue into 1-2 mm³ pieces
Sterile razor	3M	9681	Surgical prep razor, for hair removal from operative area
Sterile scalpel	Integra Miltex	4-415	Sterile disposable scalpel, for making incision
Sterile surgical scissors	Sklar	10022-580	Merit Sterile Iris Scissors, for dissecting blood vessels and connective tissue
T25 cell culture flask	Corning	430639	Falcon T25 cell culture flask, vented cap, sterile
Triton X-100 (0.1%)	Sigma-Aldrich	T8787	Laboratory grade, for permeabilization
Trypan Blue	Gibco, Thermo Fisher Scientific	15250061	0.4% solution for cell viability counting.
Trypsin-EDTA (0.25%)	Gibco	25200056	0.25% trypsin, 1 mM EDTA, for detaching ADSCs
β-Glycerophosphate	Sigma-Aldrich	G9422	Disodium salt pentahydrate, for osteogenic induction