Maximizing stem cell yield from limited adipose tissue: A novel two-step digestion approach and future artificial intelligence-integrated perspectives

Table 1 Comparison of conventional single-step and two-step digestion methods for adipose tissue-derived stromal vascular fraction isolation

Feature	Conventional single-step digestion (SVF1 only)	Two-step digestion (SVF1 + SVF2)
Total cell yield (per sample)	1.09 × 106 ± 2.07 × 105 cells	Additional 38%-40% increase per sample (SVF2 contributes 4.19 × 105 ± 1.56 × 105 cells)
Cell viability	97.22% ± 1.88%	96.84% ± 3.39% (not statistically different, P = 0.83)
Total mononuclear cells	4.11 × 105 ± 1.62 × 105 cells	SVF2 adds 1.99 × 105 ± 1.05 × 105 cells (P = 0.03)
Mononuclear cell percentage	38.71% ± 16.11%	45.46% ± 13.54% (not significantly different, P = 0.49)
Plating efficiency	0.29 ± 0.11	0.72 ± 0.34 (P = 0.03) - higher clonogenic capacity per seeded cell
CFU per 1000 mononuclear cells	8.23 ± 3.47	15.75 ± 5.50 (P = 0.03) - higher progenitor density
CD44+ cells	42.85% ± 23.21%	32.38% ± 23.55% (comparable, no significant difference)
CD73+ cells	10.86% ± 5.79%	11.41% ± 9.13% (comparable)
CD146+ cells	29.09% ± 16.23%	27.68% ± 15.21% (comparable)
Gene expression (VEGF, SDF, NOS3, CD14, MMP3, IL4R, P16, P53)	Reference expression levels	Similar transcriptional profiles; no statistically significant differences (P > 0.05)
Histological findings	After first digestion: Partial ECM degradation, moderate adipocyte disruption, some cells still embedded	After second digestion: Substantial structural breakdown, complete loss of adipocyte integrity, release of tightly bound regenerative cells
Clinical relevance	Discards residual tissue; may yield insufficient cells for pediatric or low-adipose-reserve patients	Maximizes cell harvest from small samples; particularly valuable for pediatric and low-reserve patients

SVF: Stromal vascular fraction; CFU: Colony-forming unit; VEGF: Vascular endothelial growth factor; SDF: Stromal cell-derived factor; MMP3: Matrix metalloproteinase 3; IL4R: Interleukin 4 receptor; ECM: Extracellular matrix.

Table 2 Comparison of collagenase types, digestion time, advantages, and disadvantages for isolating adipose-derived stem cells from lipoaspirate

Enzyme type	Typical concentration	Digestion time (37 °C)	Advantages	Disadvantages	Ref.
Collagenase type I	0.075%-0.2% (w/v) or 0.1-0.2 U/mL (activity-based)	30-90 minutes	Highest cell yield; optimal for loose lipoaspirate; rapid digestion; good viability	Batch-to-batch variation; animal origin; risk of over-digestion (> 60-90 minutes)	[13-15]
Collagenase type II	0.1%-0.2% (w/v) or 0.15-0.25 U/mL (activity-based)	45-90 minutes	Effective for fibrous or bloody lipoaspirate	Longer digestion; slightly lower yield compared to type I; viability may decrease (> 60 minutes)	[16,17]
Collagenase type IV	0.1%-0.2% (w/v) or 0.15-0.2 U/mL (activity-based)	45-75 minutes	Preserves surface markers (e.g., CD44, CD73); lower tryptic activity	Lower digestion efficiency (60%-80% yield of type I); longer incubation; higher cost	[18,19]
Collagenase I + dispase/thermolysin	Collagenase I: 0.075% + dispase: 0.1-0.2 U/mL	30-45 minutes	Potential for higher single-cell yield; gentler release from ECM; basis for many GMP-grade blends (e.g., liberase)	Higher cost; slight risk of over-digestion; requires pre-testing for optimal ratio	[20,21]
Recombinant/GMP-grade collagenase	As per manufacturer (activity-based)	40-60 minutes (needs calibration)	Animal-free; consistent activity; high viability; clinically safe; low endotoxin	Expensive; requires pre-testing for optimal time and concentration	[22,23]

ECM: Extracellular matrix; GMP: Good manufacturing practices.

Table 3 Comparison of different collagenase types in liposuction-related cell isolation

Collagenase type	Cell yield	Cell viability	Surface marker retention	Clinical safety	Recommended rating
Collagenase type I	★★★★★	★★★★☆	★★★★	★★	★★★★★
Collagenase type II	★★★☆	★★★	★★★	★★	★★☆
Collagenase type IV	★★★	★★★★	★★★★★	★★	★★★
Collagenase I + dispase/thermolysin	★★★★★	★★★★	★★★★	★★	★★★★
Recombinant/GMP-grade collagenase	★★★★☆	★★★★★	★★★★★	★★★★★	★★★★★

This table uses a five-star rating system to evaluate and compare different performance in each aspect. Higher star ratings indicating better performance. GMP: Good manufacturing practices.