Mechanical Micronization of Lipoaspirates for Regenerative Therapy

Summary

Here, we present a protocol to obtain stromal vascular fraction from adipose tissue through a series of mechanical processes, which include emulsification and multiple centrifugations.

Abstract

Stromal vascular fraction (SVF) has become a regenerative tool for various diseases; however, legislation strictly regulates the clinical application of cell products using collagenase. Here, we present a protocol to generate an injectable mixture of SVF cells and native extracellular matrix from adipose tissue by a purely mechanical process. Lipoaspirates are put into a centrifuge and spun at 1,200 x g for 3 min. The middle layer is collected and separated into two layers (high-density fat at the bottom and low-density fat on the top). The upper layer is directly emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance under the oil layer is collected and defined as the extracellular matrix (ECM)/SVF-gel. The oil on the top layer is collected. Approximately 5 mL of oil is added to 15 mL of high-density fat and emulsified by intersyringe shifting, at a rate of 20 mL/s for 6x to 8x. The emulsified fat is centrifuged at 2,000 x g for 3 min, and the sticky substance is also ECM/SVF-gel. After the transplantation of the ECM/SVF-gel into nude mice, the graft is harvested and assessed by histologic examination. The result shows that this product has the potential to regenerate into normal adipose tissue. This procedure is a simple, effective mechanical dissociation procedure to condense the SVF cells embedded in their natural supportive ECM for regenerative purposes.

Introduction

Stem cell therapies provide a paradigm shift for tissue repair and regeneration so that they may offer an alternative therapeutic regimen for various diseases¹. Stem cells (e.g., induced pluripotent stem cells and embryonic stem cells) have great therapeutic potential but are limited due to cell regulation and ethical considerations. Adipose-derived mesenchymal stromal/stem cells (ASCs) are easy to obtain from lipoaspirates and not subject to the same restrictions; thus, it has become an ideal cell type for practical regenerative medicine². In addition, they are nonimmunogenic and have abundant resources from au....

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. ECM/SVF-gel Preparation

1. Harvest fat.
   1. Perform liposuction on a human with a 3-mm multiport cannula, which contains several sharp side holes of 1 mm in diameter, at -0.75 atm of suction pressure.
   2. Coll....

Results

After processing the Coleman fat to ECM/SVF-gel, the volume of discarded oil takes up 80% of the final volume, and only 20% of adipose tissue preserved under the oil layer is regarded as ECM/SVF-gel (Figure 1A). ECM/SVF-gel has a smooth liquid-like texture that enables it to go through a 27 G fine needle; however, Coleman fat is comprised of an integral adipose structure with large fibers and can only go through an 18 G cannula (

Discussion

Stem cell-based regenerative therapy has shown a great potential benefit in different diseases. ASCs are outstanding therapeutic candidates because they are easy to obtain and have the capacity for tissue repair and the regeneration of novel tissues¹⁵. However, there are limitations to expanding its clinical application, since it requires complicated procedures to isolate cells and collagenase for processing⁶. Thus, it is essential to develop a simple technique to obtain st.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Alexa Fluor 488-conjugated isolectin GS-IB4	Molecular Probes	I21411
guinea pig anti-mouse perilipin	Progen	GP29
DAPI	Thermofisher	D1306
wide tip pipet	Celltreat	229211B
Confocal microscope	Leica	TCS SP2
nude nice	Southern Mdical University	/
light microscope	Olympus	/
50 mL tube	Cornig	430828
sterile bag	Laishi	/
microtome	Leica	CM1900
centrifuge	Heraus