Summary

Viable stem cells can be successfully isolated and expanded from cryopreserved microfragmented adipose tissue using both tissue explant culture and enzymatic digestion

Abstract

Background

Microfragmentation is a new non-enzymatic, mechanical one-step procedure to process stem cells from adipose tissue (AT) for usage directly in the operation theatre. Treatment of knee osteoarthritis with autologous stem cells from microfragmented AT has shown promising results. Cryopreservation and biobanking of stem cells are becoming increasingly important for research purposes, treatment of aged patients, and for repetitive treatments to improve long-term outcomes without the need for additional lipoaspirations. Isolation of viable stem cells from cryopreserved whole lipoaspirates have been described, but not from cryopreserved microfragmented AT.

Aim

To investigate if viable stem cells could be isolated and expanded from cryopreserved microfragmented AT harvested from knee osteoarthritis patients by two different isolation methods; (1) tissue explant culture (TEC), and (2) enzymatic digestion (ED).

Materials And Methods

Microfragmented subcutaneous abdominal AT from knee osteoarthritis patients was cryopreserved in cryomedium containing 10% dimethyl sulfoxide (DMSO) cryoprotectant at -80 degrees Celsius. The samples were thawed and rinsed for stem cell isolation by TEC (non-enzymatic) and ED (with 1 mg/mL collagenase type I), respectively. Viability, population doublings, and doubling time was assessed by trypan blue staining. Cell type and senescence-associated ß-galactosidase activity were measured by flow cytometry. Osteogenic and adipogenic differentiation was assessed quantitatively by Alizarin Red S and Oil-Red-O staining, respectively. Statistical analysis was performed using paired t-tests. Normality of data was confirmed using Shapiro-Wilk tests and QQ-plots. p-values <0.05 were considered statistically significant.

Results

Microfragmented AT from 7 patients (5 females and 2 males, age 41 to 63 years) was cryopreserved for a period of 46-150 days (mean (SD) 115.9 days (44.3 days)). Viable stem cells were successfully recovered and expanded from all patients using both isolation methods with no significant difference in viable population doublings or doubling time from passage 1 to 3 (p>0.05). Low levels of senescence-associated ß-galactosidase activity was detected for both methods. Stemness was verified by stem cell surface markers and osteogenic and adipogenic differentiation performance. Adventitial stem cells (CD31-/CD34+/CD45-/CD90+/CD146-), pericytes (CD31-/CD34-/CD45-/CD90+/CD146+), transitional pericytes (CD31-/CD34+/CD45-/CD90+/CD146+), mesenchymal stem cells (CD34-/CD45-/CD90+), and CD271+ stem cells (CD31-/CD45-/CD90+/CD271+) were identified using both methods. More pericytes were present when using TEC (25% (24%)) compared to ED (2% (2%)) at passage 4 (p=0.04).

Conclusion

Viable stem cells can be isolated and expanded from cryopreserved microfragmented AT using both TEC and ED. The TEC isolation method provides more clinically relevant pericytes than ED. For research purposes, the TEC method is believed to be more representative of treatment with microfragmented AT as no enzymes have been applied.