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The COVID-19 pandemic has significantly impacted the way of life worldwide and continues to bring high mortality rates to at-risk groups. Patients who develop severe COVID-19 pneumonia, often complicated with ARDS, are left with limited treatment options with no targeted therapy currently available. One of the features of COVID-19 is an overaggressive immune reaction that leads to multiorgan failure. Mesenchymal stromal cell (MSC) treatment has been in development for various clinical indications for over a decade, with a safe side effect profile and promising results in preclinical and clinical trials. Therefore, the use of MSCs in COVID-19-induced respiratory failure and ARDS was a logical step in order to find a potential treatment option for the most severe patients. In this review, the main characteristics of MSCs, their proposed mechanism of action in COVID-19 treatment and the effect of this therapy in published case reports and clinical trials are discussed.

Trehalose is a nontoxic disaccharide and a promising cryoprotection agent for medically applicable cells. In this study, the efficiency of combining trehalose with reversible electroporation for cryopreservation of two types of human mesenchymal stromal cells was investigated: adipose-derived stromal cells, and umbilical-cord-derived stromal cells. Comparable results to standard dimethyl sulfoxide cryopreservation protocols were achieved, even without extensive electroporation parameters and protocol optimization. The presence of high extracellular trehalose resulted in comparable cell viabilities without and with electroporation. According to the determination of trehalose concentrations, 250 mM extracellular trehalose resulting in, 20 mM to 50 mM intracellular trehalose were sufficient for successful cryopreservation of cells. With electroporation, higher (i.e. 50 mM to 90 mM) intracellular trehalose was achieved after cryopreservation, although cell survival was not improved significantly. To evaluate the impact of electroporation and cryopreservation on cells, stress and immune-activation-related gene expression were analyzed. Electroporation and/or cryopreservation resulted in increased SOD2 and HSPA1A expression. Despite the increased stress response, the high up-regulation by mesenchymal stromal cells of immunomodulatory genes in the inflammatory environment was not affected. Highest expression was seen for the IDO1 and TSG6 genes. In conclusion, cryopreservation of mesenchymal stromal cells in trehalose results in comparable characteristics to their cryopreservation using dimethyl sulfoxide., Competing Interests: Conflict of interestThe authors declare that they have no conflicts of interest., (© The Author(s), under exclusive licence to Springer Nature B.V. 2021.)

Background: Osteochondral injury is a very common orthopaedic pathology, mainly affecting young, active population, with limited current treatment options. Herein we are presenting cellular and early clinical data of a patient series treated for chronic osteochondral lesions in the knee with a filter-based intra-operative bone marrow aspirate (BMA) separation device., Methods: Fifteen patients with chronic knee osteochondral lesions (60% females, 19-59 years) were included in this prospective case series. Filtered BMA (f-BMA), containing mesenchymal stem/stromal cells (MSCs), was combined with a biomimetic collagen-hydroxyapatite scaffold (CHAS) and implanted into the site of the lesion. Harvested BMA and post-separation f-BMA were analysed for blood cell counts, flow cytometry, and fibroblast colony forming units (CFU-Fs). Patients were followed for serious adverse events and graft failures. Clinical evaluation was assessed using the knee injury and osteoarthritis outcome score (KOOS). In 8 patients a magnetic resonance imaging (MRI)/arthroscopy were performed., Results: Cell suspension contained 0.027% CD271 + CD45 - 7-AAD - cells, 0.15% CD73 + CD90 + CD105 + cells and 0.0012% CFU-Fs of all nucleated cells with 86% viability. Filtration process resulted in 12.8 (4.0-40.8) fold enrichment in terms of CFU-F content in comparison to initial BMA. No serious adverse events related directly to the osteochondral treatment were reported. After an average follow-up of 20 months (14-25) all KOOS subscales (Symptoms/Pain/Daily activities/Sport and recreation/Quality of life) increased significantly from pre-operative 55/56/67/30/30 to post-operative 73/76/79/51/52 (p values < 0.05), respectively. MRI or arthroscopic evaluation revealed nearly normal to normal overall International Cartilage Repair Society assessment in 7/8 patients., Conclusion: The filter-based BMA separation procedure significantly increased the frequency of mesenchymal stem/stromal cells (MSCs), however their concentration was not increased. The clinical evaluation revealed high safety profile of the treatment and resulted in improved clinical status of the patients.

Inflammation plays a major role in progression of rheumatoid arthritis, a disease treated with antagonists of tumor necrosis factor-alpha (TNF-α) and interleukin 1β (IL-1β). New in vitro testing systems are needed to evaluate efficacies of new anti-inflammatory biological drugs, ideally in a patient-specific manner. To address this need, we studied microspheroids containing 10,000 human osteoarthritic primary chondrocytes (OACs) or chondrogenically differentiated mesenchymal stem cells (MSCs), obtained from three donors. Hypothesizing that this system can recapitulate clinically observed effects of anti-inflammatory drugs, spheroids were exposed to TNF-α, IL-1β, or to supernatant containing secretome from activated macrophages (MCM). The anti-inflammatory efficacies of anti-TNF-α biologicals adalimumab, infliximab, and etanercept, and the anti-IL-1β agent anakinra were assessed in short-term microspheroid and long-term macrospheroid cultures (100,000 OACs). While gene and protein expressions were evaluated in microspheroids, diameters, amounts of DNA, glycosaminoglycans, and hydroxiproline were measured in macrospheroids. The tested drugs significantly decreased the inflammation induced by TNF-α or IL-1β. The differences in potency of anti-TNF-α biologicals at 24 h and 3 weeks after their addition to inflamed spheroids were comparable, showing high predictability of short-term cultures. Moreover, the data obtained with microspheroids grown from OACs and chondrogenically differentiated MSCs were comparable, suggesting that MSCs could be used for this type of in vitro testing. We propose that in vitro gene expression measured after the first 24 h in cultures of chondrogenically differentiated MSCs can be used to determine the functionality of anti-TNF-α drugs in personalized and preclinical studies. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:1045-1058, 2018., (© 2018 American Institute of Chemical Engineers.)

New cryopreservation approaches for medically applicable cells are of great importance in clinical medicine. Current protocols employ the use of dimethyl sulfoxide (DMSO), which is toxic to cells and causes undesirable side effects in patients, such as cardiac arrhythmias, neurological events, and others. Trehalose, a nontoxic disaccharide, has been already studied as a cryoprotectant. However, an efficient approach for loading this impermeable sugar into mammalian cells is missing. In our study, we assessed the efficiency of combining reversible electroporation and trehalose for cryopreservation of human adipose-derived stem cells. First, we determined reversible electroporation threshold by loading of propidium iodide into cells. The highest permeabilization while maintaining high cell viability was reached at 1.5 kV/cm, at 8 pulses, 100 µs, and 1 Hz. Second, cells were incubated in 250 or 400 mM trehalose and electroporated before cryopreservation. After thawing, 83.8 ± 1.8 % (mean ± SE) cell recovery was obtained at 250 mM trehalose. By using a standard freezing protocol (10 % DMSO in 90 % fetal bovine serum), cell survival after thawing was about 91.5 ± 1.6 %. We also evaluated possible effects of electroporation on cells' functionality before and after thawing. Successful cell growth and efficient adipogenic and osteogenic differentiation were achieved. In conclusion, electroporation seems to be an efficient method for loading nonpermeable trehalose into human adipose-derived stem cells, allowing long-term cryopreservation in DMSO-free and xeno-free conditions.

Background: Glioblastoma multiforme (GBM) is among the most aggressive cancers with a poor prognosis in spite of a plethora of established diagnostic and prognostic biomarkers and treatment modalities. Therefore, the current goal is the detection of novel biomarkers, possibly detectable in the blood of GBM patients that may enable an early diagnosis and are potential therapeutic targets, leading to more efficient interventions., Experimental Procedures: MicroRNA profiling of 734 human and human-associated viral miRNAs was performed on blood plasma samples from 16 healthy individuals and 16 patients with GBM, using the nCounter miRNA Expression Assay Kits., Results: We identified 19 miRNAs with significantly different plasma levels in GBM patients, compared to the healthy individuals group with the difference limited by a factor of 2. Additionally, 11 viral miRNAs were found differentially expressed in plasma of GBM patients and 24 miRNA levels significantly correlated with the patients' survival. Moreover, the overlap between the group of candidate miRNAs for diagnostic biomarkers and the group of miRNAs associated with survival, consisted of ten miRNAs, showing both diagnostic and prognostic potential. Among them, hsa miR 592 and hsa miR 514a 3p have not been previously described in GBM and represent novel candidates for selective biomarkers. The possible signalling, induced by the revealed miRNAs is discussed, including those of viral origin, and in particular those related to the impaired immune response in the progression of GBM., Conclusion: The GBM burden is reflected in the alteration of the plasma miRNAs pattern, including viral miRNAs, representing the potential for future clinical application. Therefore proposed biomarker candidate miRNAs should be validated in a larger study of an independent cohort of patients.

Background: Different immunotherapy approaches for the treatment of cancer and autoimmune diseases are being developed and tested in clinical studies worldwide. Their resulting complex experimental data should be properly evaluated, therefore reliable normal healthy control baseline values are indispensable., Methodology/principal Findings: To assess intra- and inter-individual variability of various biomarkers, peripheral blood of 16 age and gender equilibrated healthy volunteers was sampled on 3 different days within a period of one month. Complex "crossomics" analyses of plasma metabolite profiles, antibody concentrations and lymphocyte subset counts as well as whole genome expression profiling in CD4+T and NK cells were performed. Some of the observed age, gender and BMI dependences are in agreement with the existing knowledge, like negative correlation between sex hormone levels and age or BMI related increase in lipids and soluble sugars. Thus we can assume that the distribution of all 39.743 analysed markers is well representing the normal Caucasoid population. All lymphocyte subsets, 20% of metabolites and less than 10% of genes, were identified as highly variable in our dataset., Conclusions/significance: Our study shows that the intra-individual variability was at least two-fold lower compared to the inter-individual one at all investigated levels, showing the importance of personalised medicine approach from yet another perspective.

Basic functional test for evaluation of in vitro cultured human dendritic cells (DC) is primary allogeneic one-way mixed lymphocyte reaction (MLR). In this way, one can evaluate stimulating capacity, which is a basic characteristic of DC. The proliferation of cells is measured through incorporation of 3 H-thymidine. Normally proliferation is measured at days 5-7. We studied kinetics of proliferative responses initiated with different stimulating cell suspensions to evaluate differences and possibly reduce time needed to perform this test.Gradual increase in response from days 1 to 7 and a significant difference from controls (peripheral blood mononuclear cells) seen from day 4 was noted if macrophages were used as stimulators. A consistently higher proliferation, compared to controls, was always found already on day 2 when mature DC were used as stimulators. The reaction peaked 2 to 3 days earlier and was also more than two times more intense.This maximal and significantly higher response, consistently seen already after 48 hours, allows us to confirm the presence of mature DC in stimulating suspensions much earlier than previously.

Chondrocytes present in articular cartilage survive as a resident cell population throughout the lifespan of the individual organism. However, articular chondrocytes as other cells also undergo apoptosis and there is an ever increasing list of diverse stimuli that can induce this phenomenon in vitro. Our main interest was to investigate potential cytotoxic effects of vitamin C (L-ascorbic acid) on human articular chondrocytes. The present study suggests that vitamin C can induce apoptosis in a cell culture of chondrocytes after 18 h of cultivation. Apoptosis-inducing activity of L-ascorbic acid is dose dependent and significantly affected by the presence of serum. The increased number of vitamin C induced apoptotic cells was associated with DNA fragmentation and morphological changes of the cells.