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

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.