Successful isolation of viable stem cells from cryopreserved microfragmented human adipose tissue from patients with knee osteoarthritis – a comparative study of isolation by tissue explant culture and enzymatic digestion

Abstract

Purpose To investigate if viable stem cells could be isolated and expanded from cryopreserved microfragmented adipose tissue (AT) harvested from patients with knee osteoarthritis. Methods Microfragmented abdominal AT from knee osteoarthritis patients was cryopreserved at -80 °C in cryoprotectant-medium. The samples were thawed for stem cell isolation by tissue explant culture (TEC) and enzymatic digestion (ED), respectively. Viability, population doublings, and doubling time were assessed by trypan blue staining and flow cytometry. Cell type and senescence-associated β-galactosidase activity were analyzed by flow cytometry. Osteogenic and adipogenic differentiation was assessed quantitatively by Alizarin-Red-S and Oil-Red-O staining, respectively. Results Microfragmented AT from 7 patients 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 β-galactosidase activity was detected for both methods with no significant difference between TEC and ED (p = 0.17). 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+), 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 (3% (2%)) at passage 4 (p = 0.04). Conclusions Viable stem cells can be isolated and expanded from cryopreserved microfragmented AT using both TEC and ED. TEC provides more clinically relevant pericytes than ED.

Microfragmented abdominal adipose tissue‐derived stem cells from knee osteoarthritis patients aged 29–65 years demonstrate in vitro stemness and low levels of cellular senescence

“…However, Ragni et al [ 39 ] only assessed senescence using this specific parameter and used enzymes for processing which differs from the current study where tissue explants were used to represent the non‐enzymatic processing of clinically used ADSCs from MF AT. No apparent difference was found between the level of senescence in samples from frozen MF AT and samples from MF AT directly cultivated as tissue explants [ 4 ].…”

“…Seven of the MF lipoaspirates were directly cryopreserved for 46–150 days as described in Bagge et al [ 4 ] until the start of tissue explant culture isolation and cultivation. The remaining samples ( n = 13) were isolated by tissue explant cultivation immediately after harvesting according to the procedure described in Bagge et al [ 4 ].…”

“…Flow cytometry was used to analyse the immunophenotype of the MF‐ADSCs at passage 4 according to the manufacturer's protocol using multiple anti‐human fluorescent primary antibodies; CD31‐FITC, CD34‐APC, CD45‐BV786, CD90‐PE, CD146‐BV421 and CD271‐PE‐CyTM7 (BD Biosciences, Horizon subtype, mouse IgG 1 κ isotype) and analysed on a flow cytometer (BD LSR Fortessa with BD FACSDiva 8.0.3 software) as described by Bagge et al [ 4 ]. Flow cytometry data was analysed using BD FACSDiva Software 8.0.3 (Figure 1a–g ).…”

“…The remaining wells were kept as non‐induced controls and grown in AT expansion medium throughout the entire 14 and 21 days of the differentiation protocols, respectively. The cells were quantitatively stained with Oil‐Red‐O (Sigma‐Aldrich, Cat# O0625) to assess adipogenesis and Alizarin Red S (ARed‐Q, ScienCell, Cat# 8678) to assess osteogenesis, as described by Bagge et al [ 4 ].…”

New characterization and safety evaluation of human limbal stem cells used in clinical application: fidelity of mitotic process and mitotic spindle morphologies

“…The calculation of cumulative population doubling (cPDs) and doubling time (DT) was performed when cells reached 80–90% confluency as follows: where N is the number of live cells trypsinized in subconfluence, N 0 is the initial number of live cells seeded, and CT is the total number of culture days [ 25 ].…”