For cell‐based assays studying monocytes and macrophages, the immortalized monocyte cell line THP‐1 is widely used and stimulated with phorbol 12‐myristate 13‐acetate, lipopolysaccharide (LPS) and/or interferon‐γ (IFN‐γ), after which it differentiates and polarizes into proinflammatory M1‐like macrophages. For the quantification of this and the effect of different factors affecting these processes, the expression levels of various maturation markers are determined using reverse transcription‐quantitative PCR. For this purpose, stably expressed reference genes are crucial. However, no studies evaluating the stability of reference genes in THP‐1 cells stimulated with LPS and IFN‐γ have been performed. Therefore, this paper describes the selection of the most used reference genes [RPL37A (ribosomal protein L37a), GAPDH (glyceraldehyde‐3‐phosphate dehydrogenase), UBC (ubiquitin C), B2M (0β2‐microbulin), ACTB (β‐actin) and PPIA (cyclophilin A)], the in silico primer design, the analysis and the validation of these in accordance with the MIQE (Minimum Information for Publication of Quantitative Real‐Time PCR Experiments) guidelines and more recent recommendations for the validation of the stability of reference genes. Using the RefFinder platform, including the four most popular algorithms for reference gene validation, the Delta CT, BestKeeper, NormFinder and geNorm, we find the reference genes GAPDH and UBC to be the most stable. Furthermore, we demonstrate that the normalization of gene expression data using the least stable reference genes, ACTB and B2M, dramatically affects the interpretation of experimental data. Taken together, it is vital to validate the stability of reference genes under the specific experimental conditions used when utilizing the THP‐1 monocyte model system.

The exploration of multi-dimensional brain activity with high temporal and spatial resolution is of great significance in the diagnosis of neurological disease and the study of brain science. Although the integration of electroencephalogram (EEG) with magnetic resonance imaging (MRI) and computed tomography (CT) provides a potential solution to achieve a brain-functional image with high spatiotemporal resolution, the critical issues of interface stability and magnetic compatibility remain challenging. Therefore, in this research, we proposed a conductive hydrogel EEG electrode with an asymmetrical bilayer structure, which shows the potential to overcome the challenges. Benefiting from the bilayer structure with different moduli, the hydrogel electrode exhibits high biological and mechanical compatibility with the heterogeneous brain-electrode interface. As a result, the impedance can be reduced compared with conventional metal electrodes. In addition, the hydrogel-based ionic conductive electrodes, which are free from metal conductors, are compatible with MRI and CT. Therefore, they can obtain high spatiotemporal resolution multi-dimensional brain information in clinical settings. The research outcome provides a new approach for establishing a platform for early diagnosis of brain diseases and the study of brain science.

In pre-clinical studies, human adipose-derived stem cells (hASCs) have shown great promise as a treatment modality for healing of cutaneous wounds. The advantages of hASCs are that they are relatively easy to obtain in large numbers from basic liposuctions, they maintain their characteristics after long-term in vitro culture, and they possess low immunogenicity, which enables the use of hASCs from random donors. It has been hypothesized that hASCs exert their wound healing properties by reducing inflammation, inducing angiogenesis, and promoting fibroblast and keratinocyte growth. Due to the inherent variability associated with the donor-dependent nature of ASC-based products, it appears necessary that the quality of the different products is prospectively certified using a set of most relevant potency assays. In this review, we present an overview of the available methodologies to assess the Mode and the Mechanism of Action of hASCs, specifically in the wound healing scenario. In conclusion, we propose a panel of potential potency assays to include in the future production of ASC-based medicinal products.

In order to enhance the therapeutic potential, it is important that sufficient knowledge regarding the dynamic changes of adipose-derived stem cell (ASC) immunophenotypical and biological properties during in vitro growth is available. Consequently, we embarked on a study to follow the evolution of highly defined cell subsets from three unrelated donors in the course of eight passages on tissue culture polystyrene. The co-expression patterns were defined by panels encompassing seven and five cell surface markers, including CD34, CD146, CD166, CD200, CD248, CD271, and CD274 and CD29, CD31, CD36, CD201, and Stro-1, respectively. The analysis was performed using multichromatic flow cytometry. We observed a major paradigm shift, where the CD166-CD34⁺ combination which was found across all cell subsets early in the culture was replaced by the CD166⁺ phenotype as the population homogeneity increased with time. At all analysis points, the cultures were dominated by a few major clones that were highly prevalent in most of the donors. The selection process resulted in two predominant clones in the larger panel (CD166⁺CD34⁻CD146⁻CD271⁻ CD274⁻CD248⁻CD200⁻ and CD166⁺CD34⁺ CD146⁻CD271⁻CD274⁻CD248⁻CD200⁻) and one clone in the smaller panel (CD29⁺CD201⁺CD36⁻ Stro-1⁻ CD31⁻). The minor subsets, including CD166⁺CD34⁻CD146⁻CD271⁺CD274⁻CD248⁻CD200⁻ and CD166⁺CD34⁺CD146⁺CD271⁻CD274⁻CD248⁻CD200⁻, and CD29⁺CD201⁻CD36⁻Stro-1⁻CD31⁻, CD29⁺CD201⁺CD36⁻Stro-1⁺CD31⁻, and CD29⁺CD201⁺CD36⁺Stro-1⁻CD31⁻, in the seven and five marker panels, respectively, were, on the other, hand highly fluctuating and donor-dependent. The results demonstrate that only a limited number of phenotypical repertoires are possible in ASC cultures. Marked differences in their relative occurrence between distinct individuals underscore the need for potency standardization of different ASC preparation to improve the clinical outcome.

It has been suggested that immunophenotypically defined lineages within the in vitro expanded adipose-derived stem cell (ASC) may play a beneficial role from the perspective of a personalized intervention. Therefore, to better understand the implications of different surface marker profiles for the functionality, we set out to examine the evolution of ASC-variants based on the co-expression of five bright or eight dim epitopes. At passages P1, P4, and P8, the co-localization of five bright markers (CD73, CD90, CD105, CD166, and CD201), or eight dim markers (CD34, CD36, CD200, CD248, CD271, CD274, CD146, and the Stro-1), was investigated by flow cytometry. Selected subpopulations were isolated using the fluorescence-activated cells sorting from the cryopreserved P4 and analyzed in terms of proliferative and clonogenic properties, trilineage differentiation, and wound healing potential. Only two of the dim epitopes were found in representative subpopulations (SP), and from the P4 onwards, two major combinations featuring the CD274⁺ (SP1) or the CD274⁺ CD146⁺ (SP2) emerged. Upon sorting and growth, both subpopulations assumed new but highly similar clonal profiles, consisting of the CD274⁺ CD146⁺ and the CD274⁺ CD146⁺ CD248⁺ phenotypes. The functional analysis revealed that the SP2 surpassed SP1 and the unfractionated cells regarding the growth rate, clonogenic activity, and the wound closure and endothelial tube formation potential. The surface epitopes may be considered a tool to enrich specific functionality and thus improve therapeutic outcomes in dedicated circumstances.

Cryopreservation enhances the availability of “off-the-shelf” cell therapies. However, the choice between tissue culture polystyrene (TCP) and hollow fiber system (HFB) system for adipose-derived stem cell (ASC) production remains a critical decision, with implications for scalability, reproducibility, and the clinical efficacy. Therefore, the characteristics of ASCs expanded in TCP and HFB and cryopreserved were compared. TCP and HFB cultures were established, and cells were cryopreserved. Surface markers were analyzed to identify immunophenotypic changes and subpopulations. Clonogenicity, differentiation capability, and proliferation potentials were determined along with surrogate tests on wound healing. The expressions of the most markers were consistent before and after thawing for both systems. However, CD105 expression of TCP cells was significantly decreased by the freeze-thawing procedure. Also, CD274 was significantly less expressed on HFB-expanded cells before freezing, however, post-thawing, the proportion of CD274 positive cells was comparable to TCP cells. Besides, two expansions supported different subpopulations, influencing the heterogeneity within ASC cultures. Despite this heterogeneity, no statistical differences were observed as for ASC functional characteristics and the effects on fibroblasts. This study highlighted freeze-thaw does not interfere with the production of fully functional ASCs in either system, although it drives some differential changes in the subpopulations between systems.