Inducing Heat Shock Proteins Enhances the Stemness of Frozen–Thawed Adipose Tissue-Derived Stem Cells

Abstract

Extensive research has been performed to determine the effect of freezing protocol and cryopreservation agents on the viability of adipose tissue-derived stromal/stem cells (ASCs) as well as other cells. Unfortunately, the conclusion one may draw after decades of research utilizing fundamentally similar cryopreservation techniques is that a barrier exists, which precludes full recovery. We hypothesize that agents capable of inducing a subset of heat shock proteins (HSPs) and chaperones will reduce the intrinsic barriers to the post-thaw recovery of ASCs. ASCs were exposed to 43°C for 1 h to upregulate HSPs, and the temporal HSP expression profile postheat shock was determined by performing quantitative polymerase chain reaction (PCR) and western blotting assays. The expression levels of HSP70 and HSP32 were found to be maximum at 3 h after the heat shock, whereas HSP90 and HSP27 remain unchanged. The heat shocked ASCs cryopreserved during maximal HSPs expression exhibited increased post-thaw viability than the nonheat shocked samples. Histochemical staining and quantitative reverse transcription-PCR indicated that the ASC differentiation potential was retained. Thus, suggesting that the upregulation of HSPs before a freezing insult is beneficial to ASCs and a potential alternative to the use of harmful cryoprotective agents.

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

“…For control samples, stromal media containing DMEM with 10% FBS and 1% antibiotic was used. After 10 days, the adipogenic differentiated cultures were fixed in 4% paraformaldehyde and stained using the Oil Red O stain as described in several previous studies 17 , 19 , 21 , 22 . Quantitation of adipogenic staining was carried out by eluting the Oil Red O stain by 100% isopropanol followed by measuring absorbance of the elution at 510 nm.…”

“…Similarly, ASCs cryopreserved with DMSO and PVP and stored for 2 weeks at −196 °C maintained their osteogenic and adipogenic potentials as well as their post thaw viability 21 , 39 . We have recently reported that thermally pre-conditioned ASCs cryopreserved in either DMSO or PVP and stored for 1 day at −196 °C displayed enhanced cell viability while retaining their differentiation plasticity 22 . Cryopreservation of human ASCs for 3 months in liquid nitrogen displayed no sign of deterioration in their functionality regarding immunophenotype, differentiation potential, proliferation, and viability 67 .…”

“… 30 . Further studies are clearly needed to evaluate whether the reduced osteogenic potential of the long-term cryopreserved ASCs can be enhanced if the post-thaw media is supplemented with additional growth factors or by thermal pre-conditioning of cell culture 22 .…”

Hybrid Synthetic‐Biological Hydrogel System for Adipose Tissue Regeneration

“…For Oil Red O staining, the samples were fixed in a 4% paraformaldehyde solution for 10 min, washed with PBS and stained with 0.3% w/v% Oil Red O solution (in 60% isopropanol) for 8 min, followed by washing with 60% isopropanol for 30 s. Bright field images were taken by microscope (IX73, Olympus, USA). The samples were then de‐stained with 100% isopropanol for 5 min to extract the Oil Red O according to the previous study . Absorbance of the de‐staining solution was measured at 510 nm wavelength using a plate reader (SpectraMax M5, Molecular Devices LLC, USA).…”

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.…”