Effects of Decade Long Freezing Storage on Adipose Derived Stem Cells Functionality

Abstract

Over the last decade and half, the optimization of cryopreservation for adipose tissue derived stromal/stem cells (ASCs) especially in determining the optimal combination of cryoprotectant type, cooling rate, and thawing rate have been extensively studied. In this study, we examined the functionality of ASCs that have been frozen-stored for more than 10 years denoted as long-term freezing, frozen within the last 3 to 7 years denoted as short-term freezing and compared their response with fresh ASCs. The mean post-thaw viability for long-term frozen group was 78% whereas for short-term frozen group 79% with no significant differences between the two groups. The flow cytometry evaluation of stromal surface markers, CD29, CD90, CD105, CD44, and CD73 indicated the expression (above 95%) in passages P1-P4 in all of the frozen-thawed ASC groups and fresh ASCs whereas the hematopoietic markers CD31, CD34, CD45, and CD146 were expressed extremely low (below 2%) within both the frozen-thawed and fresh cell groups. Quantitative real time polymerase chain reaction (qPCR) analysis revealed some differences between the osteogenic gene expression of long-term frozen group in comparison to fresh ASCs. Intriguingly, one group of cells from the short-term frozen group exhibited remarkably higher expression of osteogenic genes in comparison to fresh ASCs. The adipogenic differentiation potential remained virtually unchanged between all of the frozen-thawed groups and the fresh ASCs. Long-term cryopreservation of ASCs, in general, has a somewhat negative impact on the osteogenic potential of ASCs, especially as it relates to the decrease in osteopontin gene expression but not significantly so with respect to RUNX2 and osteonectin gene expressions. However, the adipogenic potential, post thaw viability, and immunophenotype characteristics remain relatively intact between all the groups.

Multimodal Label-free Monitoring of Adipogenic Stem Cell Differentiation using Endogenous Optical Biomarkers

“…The PBS solution were warmed to 37 o C and the washing steps were repeated four times. Detailed cell isolation and culture processing procedures are available elsewhere [7,40]. After isolation, the cells were centrifuged at 300g for 5 min.…”

“…Human adipose derived stem cells (hASCs) have been well established as a potential tool for tissue engineering and regenerative medicine applications due to their easy access and lack of ethical issues associated with embryonic stem cells. The hASCs properties of self-renewal, multipotency with respect to differentiation along various cell lineages, and the ability to secrete cytokines and chemokines have been extensively reported during the past decade [4][5][6][7]. Adipogenic stem-cell culture requires differentiation times ranging from 9 to 21 days and is expensive.…”

Fundamentals of Stem Cells: Why and How Patients' Own Adult Stem Cells Are the Next Generation of Medicine

“…It is of note that SPARC is also expressed by ASCs in vitro (Shaik et al, 2018). Moreover, the SPARC-related modular calcium-binding protein 1 (SMOC1), a member of the SPARC family and serving as a regulator of osteoblast differentiation, was found in the secretome of bone marrow derived MSCs (Choi et al, 2010).…”

Osteogenic Differentiation of Adipose-Derived Stromal Cells: From Bench to Clinics

“…10 It was also shown that their long-term cryopreservation for more than 10 years does not impact their viability, differentiation potential, and immunophenotype characteristics. 11 Importantly, having demonstrated their capacity for multilineage differentiation, including adipogenic, chondrogenic, myogenic, and osteogenic lineages, 3 ASCs have been used in vivo to regenerate fat, 12 cartilage 13 and bone, 14,15 and more complex tissues such as periodontium 16 and intervertebral disc. 17 Since they can also differentiate into endothelial cells, ASCs may serve as a single-cell source for the engineering of vascularized bone tissues, 18,19 thus tackling a major bottleneck in the clinical translation of tissue engineering strategies.…”