# An Experimental Sequential Digestion Method for Efficient Isolation of Human Adipose-Derived Microvascular Fragments with Enhanced Angiogenic Potential

## TL;DR

A new sequential digestion method improves the isolation of high-quality microvascular fragments from human fat, boosting their potential for tissue regeneration.

## Contribution

A novel sequential enzymatic digestion protocol for isolating high-quality human adipose-derived microvascular fragments.

## Key findings

- The sequential protocol increased MVF yield by 2.2-fold compared to conventional methods.
- MVF viability was significantly higher (93.3% vs. 75.6%) with better structural integrity.
- Sequentially isolated MVFs showed enhanced angiogenic performance in vitro and in vivo.

## Abstract

Microvascular fragments (MVFs) are intact vascular segments derived from adipose tissue that possess considerable potential for promoting tissue vascularization in regenerative medicine. However, conventional single-step enzymatic digestion methods often lead to incomplete adipose tissue dissociation and poor MVF quality.

We developed and validated a sequential enzymatic digestion protocol optimized for isolating MVFs from human lipoaspirate. Adipose samples were processed using either a conventional one-step collagenase digestion or a three-step sequential method. MVFs were evaluated for yield, viability, structural integrity, cellular phenotype, and angiogenic function both in vitro and in vivo.

Compared with the conventional approach, the sequential protocol produced a 2.2-fold increase in MVF yield and significantly reduced undigested tissue residues (p < 0.0001). MVFs isolated by the sequential protocol showed superior cell viability (93.3% vs. 75.6%), a greater proportion of long fragments, preserved endothelial and perivascular architecture, and enhanced angiogenic performance in collagen gel assays and mouse subcutaneous implantation models.

This optimized sequential digestion protocol enables the efficient and producible isolation of high-quality MVFs from human adipose tissue. It holds great promise for applications in vascularized tissue engineering and regenerative therapies.

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