Potential of animal-welfare compliant and sustainably sourced serum from pig slaughter blood.

The animal product most used as a stimulatory additive for cell cultivation is still fetal bovine serum (FBS). Besides the ethical concerns regarding serum collection, the main problems of FBS are batch-to-batch variability and the resulting risk of lower reproducibility, the differences between species, the presence of undefined/unknown components, and the risk of contamination. In contrast, pig blood, which is a by-product of slaughter, is a sufficiently available and sustainable resource with a high degree of standardization in terms of donor age, weight, and genetics. The variations in preparations from pig slaughter blood seem to be comparatively low, and consequently, batch effects might be much smaller, suggesting that the reproducibility of the research data obtained may be increased. Our pilot study aimed to investigate, as a proof of concept, whether adult human and porcine stem cells of different tissue origins proliferate and differentiate adequately when FBS is completely or partially replaced by porcine serum (PS). We could show that the human and porcine stem cells were vital and proliferated under partial and full PS supplementation. Furthermore, using PS, the two cell types studied showed tissue-specific differentiation (i.e., lipid vacuoles as a sign of adipogenic or myotubes as a sign of myogenic differentiation). In conclusion, the pig slaughter blood-derived serum has promising potential to be a replacement for FBS in adult stem cell cultures. Therefore, it could serve as a basis for the development of new cell culture supplements.

Impact of Metal Ions on Cellular Functions: A Focus on Mesenchymal Stem/Stromal Cell Differentiation.

Metals play a crucial role in the human body, especially as ions in metalloproteins. Essential metals, such as calcium, iron, and zinc are crucial for various physiological functions, but their interactions within biological networks are complex and not fully understood. Mesenchymal stem/stromal cells (MSCs) are essential for tissue regeneration due to their ability to differentiate into various cell types. This review article addresses the effects of physiological and unphysiological, but not directly toxic, metal ion concentrations, particularly concerning MSCs. Overloading or unbalancing of metal ion concentrations can significantly impair the function and differentiation capacity of MSCs. In addition, excessive or unbalanced metal ion concentrations can lead to oxidative stress, which can affect viability or inflammation. Data on the effects of metal ions on MSC differentiation are limited and often contradictory. Future research should, therefore, aim to clarify the mechanisms by which metal ions affect MSC differentiation, focusing on aspects such as metal ion interactions, ion concentrations, exposure duration, and other environmental conditions. Understanding these interactions could ultimately improve the design of biomaterials and implants to promote MSC-mediated tissue regeneration. It could also lead to the development of innovative therapeutic strategies in regenerative medicine.

Cold Atmospheric Pressure Plasma-Activated Medium Modulates Cellular Functions of Human Mesenchymal Stem/Stromal Cells In Vitro.

Cold atmospheric pressure plasma (CAP) offers a variety of therapeutic possibilities and induces the formation of reactive chemical species associated with oxidative stress. Mesenchymal stem/stromal cells (MSCs) play a central role in tissue regeneration, partly because of their antioxidant properties and ability to migrate into regenerating areas. During the therapeutic application, MSCs are directly exposed to the reactive species of CAP. Therefore, the investigation of CAP-induced effects on MSCs is essential. In this study, we quantified the amount of ROS due to the CAP activation of the culture medium. In addition, cell number, metabolic activity, stress signals, and migration were analyzed after the treatment of MSCs with a CAP-activated medium. CAP-activated media induced a significant increase in ROS but did not cause cytotoxic effects on MSCs when the treatment was singular and short-term (one day). This single treatment led to increased cell migration, an essential process in wound healing. In parallel, there was an increase in various cell stress proteins, indicating an adaptation to oxidative stress. Repeated treatments with the CAP-activated medium impaired the viability of the MSCs. The results shown here provide information on the influence of treatment frequency and intensity, which could be necessary for the therapeutic application of CAP.

Oxidative Stress Response in Adipose Tissue-Derived Mesenchymal Stem/Stromal Cells.

Reactive oxygen species (ROS) can irreversibly damage biological molecules, a process known as oxidative stress. Elevated ROS levels are associated with immune cell activation. Sustained immune system activation can affect many different cells in the environment. One cell type that has been detected in almost all tissues of the body is mesenchymal stem/stromal cells (MSC). MSC possess proliferation and differentiation potential, thus facilitating regeneration processes. However, the regenerative capacity of MSC might be impaired by oxidative stress, and the effects of long-term oxidative stress on MSC functions are sparsely described. The examination of oxidative stress is often performed by exposure to H2O2. Since H2O2 is rapidly degraded, we additionally exposed the cell cultures to glucose oxidase (GOx), resulting in sustained exposure to H2O2. Using these model systems, we have focused on the effects of short- and long-term oxidative stress on viability, migration, differentiation, and signaling. All cellular functions examined were affected by the applied oxidative stress. The differences that occur between pulsed and sustained oxidative stress indicated higher oxidative stress in MSC upon direct H2O2 exposure, whereas the GOx-induced prolonged exposure to H2O2 seems to allow for better cellular adaptation. The mechanisms underlying these different responses are currently unknown.

In vivo vaccination with cell line-derived whole tumor lysates: neoantigen quality, not quantity matters.

BACKGROUND: Cancer vaccines provide a complex source of neoantigens. Still, increasing evidence reveals that the neoantigen quality rather than the quantity is predictive for treatment outcome. METHODS: Using the preclinical Mlh1-/- tumor model, we performed a side-by side comparison of two autologous cell-line derived tumor lysates (namely 328 and A7450 T1 M1) harboring different tumor mutational burden (TMB; i.e. ultra-high: 328; moderate-high: A7450 T1 M1). Mice received repetitive prophylactic or therapeutic applications of the vaccine. Tumor incidence, immune responses and tumor microenvironment was examined. RESULTS: Both tumor cell lysates delayed tumor formation in the prophylactic setting, with the A7450 T1 M1 lysate being more effective in decelerating tumor growth than the 328 lysate (median overall survival: 37 vs. 25 weeks). Comparable results were achieved in therapeutic setting and could be traced back to antigen-driven immune stimulation. Reactive T cells isolated from A7450 T1 M1-treated mice recognized autologous Mlh1-/- tumor cells in IFNγ ELISpot, but likewise YAC-1 cells, indicative for stimulation of both arms of the immune system. By deciphering local effects, vaccines shaped the tumor microenvironment differently. While A7450 T1 M1 prophylactically vaccinated tumors harbored low numbers of myeloid-derived suppressor cells (MDSC) and elevated CD8-T cell infiltrates, vaccination with the 328 lysate evoked MDSC infiltration. Similar effects were seen in the therapeutic setting with stable disease induction only upon A7450 T1 M1 vaccination. Untangling individual response profiles revealed strong infiltration with LAG3+ and PD-L1+ immune cells when treatments failed, but almost complete exclusion of checkpoint-expressing lymphocytes in long-term survivors. CONCLUSIONS: By applying two tumor cell lysates we demonstrate that neoantigen quality outranks quantity. This should be considered prior to designing cancer vaccine-based combination approaches.

Investigation of the Effect of High Shear Stress on Mesenchymal Stem Cells Using a Rotational Rheometer in a Small-Angle Cone-Plate Configuration.

Within the healthy human body, cells reside within the physiological environment of a tissue compound. Here, they are subject to constant low levels of mechanical stress that can influence the growth and differentiation of the cells. The liposuction of adipose tissue and the subsequent isolation of mesenchymal stem/stromal cells (MSCs), for example, are procedures that induce a high level of mechanical shear stress. As MSCs play a central role in tissue regeneration by migrating into regenerating areas and driving regeneration through proliferation and tissue-specific differentiation, they are increasingly used in therapeutic applications. Consequently, there is a strong interest in investigating the effects of shear stress on MSCs. In this study, we present a set-up for applying high shear rates to cells based on a rotational rheometer with a small-angle cone-plate configuration. This set-up was used to investigate the effect of various shear stresses on human adipose-derived MSCs in suspension. The results of the study show that the viability of the cells remained unaffected up to 18.38 Pa for an exposure time of 5 min. However, it was observed that intense shear stress damaged the cells, with longer treatment durations increasing the percentage of cell debris.

T cells that target carcinoembryonic antigen eradicate orthotopic pancreatic carcinomas without inducing autoimmune colitis in mice.

BACKGROUND & AIMS: New treatment approaches are needed for patients with pancreatic adenocarcinoma. Carcinoembryonic antigen (CEA) is highly expressed on the surface of pancreatic adenocarcinoma cells; we investigated the effects of cytolytic T cells that recognize CEA in a mouse model of pancreatic carcinoma. METHODS: Immune-competent mice that expressed the CEA transgene (CEAtg) in the intestinal and pulmonary tracts were given intrapancreatic injections of Panc02 CEA(+) cells (express CEA and click beetle luciferase) and tumors were grown for 10 days. Mice were then given single intravenous injections of T cells engineered to express a chimeric antigen receptor (CAR) with high specificity, but moderate affinity, for CEA and a luminescence marker. RESULTS: Injection of the anti-CEA CAR T cells reduced the size of pancreatic tumors to below the limit of detection in all mice and produced long-term tumor eradication in 67% of mice. T cells also eradicated CEA(+) fibrosarcoma cells injected 45 days later. Bioluminescence imaging revealed the accumulation and persistence of the T cells at the tumor site. The efficacy of the T cells did not require lymphodepletion and was not reduced by soluble CEA. Mice developed some noninflammatory infiltrations of CAR(+) T cells in intestine and lung, but there was no evidence of destruction of CEA(+) healthy tissues. CONCLUSIONS: Injection of T cells that target CEA can eradicate tumors grown from CEA(+) pancreatic carcinoma cells in the pancreas of CEAtg mice without autoimmune effects.

3D Spheroid Cultivation Alters the Extent and Progression of Osteogenic Differentiation of Mesenchymal Stem/Stromal Cells Compared to 2D Cultivation.

Mesenchymal stem/stromal cells (MSC) are capable of progenitor cell fraction renewal or tissue-specific differentiation. These properties are maintained during in vitro cultivation, making them an interesting model system for testing biological and pharmacological compounds. Cell cultivation in 2D is commonly used to study cellular responses, but the 2D environment does not reflect the structural situation of most cell types. Therefore, 3D culture systems have been developed to provide a more accurate physiological environment in terms of cell-cell interactions. Since knowledge about the effects of 3D culture on specific differentiation processes is limited, we studied the effects on osteogenic differentiation and the release of factors affecting bone metabolism for up to 35 days and compared them with the effects in 2D culture. We demonstrated that the selected 3D model allowed the rapid and reliable formation of spheroids that were stable over several weeks and both accelerated and enhanced osteogenic differentiation compared with the 2D culture. Thus, our experiments provide new insights into the effects of cell arrangement of MSC in 2D and 3D. However, due to the different culture dimensions, various detection methods had to be chosen, which in principle limits the explanatory power of the comparison between 2D and 3D cultures.

Dose-Dependent Effects of Platelet-Rich Plasma Powder on Chondrocytes In Vitro.

BACKGROUND: Platelet-rich plasma (PRP) is widely used in sports medicine. However, neither preparation nor parameters for clinical application, such as concentration, timing, and number of applications, are standardized, making research and clinical utilization challenging. PURPOSE: To investigate the effect of varying doses of PRP powder in terms of different concentrations, timing, and number of applications on human chondrocytes in a reproducible cell culture model. STUDY DESIGN: Controlled laboratory study. METHODS: A standardized lyophilized platelet growth factor preparation (PRP powder) was used to stimulate human chondrocytes. Chondrocytes were cultivated for 2 weeks with different stimulation frequencies (2×, 3×, 6×) and different concentrations of PRP powders (0.5%, 1%, 5%). Cell proliferation and metabolic cell activity were analyzed on days 7 and 14. Phenotypic changes were visualized through live-dead staining. Chondrogenic differentiation was quantified with enzyme-linked immunosorbent assay to assess the synthesis of procollagen types 1 and 2. Furthermore, sulfated proteoglycans and glycosaminoglycans were analyzed. RESULTS: Human chondrocytes exhibited a significant dose- and time-dependent increase after 14 days in cell number (1% and 5% PRP powder vs unstimulated control: 7.95- and 15.45-fold increase, respectively; 2× vs 6× stimulation with 5% PRP powder: 4.00-fold increase) and metabolic cell activity (1% and 5% PRP powder vs unstimulated control: 3.27-fold and 3.58-fold change, respectively). Furthermore, cells revealed a significant increase in the amount of bone-specific procollagen type 1 (14 days, 1.94-fold) and sulfated glycosaminoglycans (14 days, 2.69-fold); however, no significant change was observed in the amount of cartilage-specific collagen type 2. CONCLUSION: We showed that chondrocytes exhibit a significant dose- and time-dependent increase in cell number and metabolic cell activity. The standardized use of growth factor concentrates in cell culture models can contribute to clinical knowledge in terms of dosage and timing of PRP applications. CLINICAL RELEVANCE: Problems with PRP, such as the absence of standardization, lack of consistency among studies, and unknown dosage, could be solved by using characterized PRP powder made by pooling and lyophilizing multiple platelet concentrates. The innovative PRP powder generates new possibilities for PRP research, as well as for the treatment of patients.

TGF-ß1 Induces Changes in the Energy Metabolism of White Adipose Tissue-Derived Human Adult Mesenchymal Stem/Stromal Cells In Vitro.

Adipose tissue plays an active role in the regulation of the body´s energy balance. Mesenchymal stem/stromal cells from adipose tissue (adMSC) are the precursor cells for repair and adipogenesis. Since the balance of the differentiation state of adipose tissue-resident cells is associated with the development of various diseases, the examination of the regulation of proliferation and differentiation of adMSC might provide new therapeutic targets. Transforming growth factor-ß1 (TGF-ß1) is synthetized by many cell types and is involved in various biological processes. Here, we investigated the effects of different concentrations of TGF-ß1 (1-10 ng/mL) on adMSC proliferation, metabolic activity, and analyzed the gene expression data obtained from DNA microarrays by bioinformatics. TGF-ß1 induced the concentration- and time-dependent increase in the cell number of adMSC with simultaneously unchanged cell cycle distributions. The basal oxygen consumption rates did not change significantly after TGF-ß1 exposure. However, glycolytic activity was significantly increased. The gene expression analysis identified 3275 differentially expressed genes upon exposure to TGF-ß1. According to the pathway enrichment analyses, they also included genes associated with energy metabolism. Thus, it was shown that TGF-ß1 induces changes in the energy metabolism of adMSC. Whether these effects are of relevance invivo and whether they contribute to pathogenesis should be addressed in further examinations.

RNA-Based Strategies for Cardiac Reprogramming of Human Mesenchymal Stromal Cells.

Multipotent adult mesenchymal stromal cells (MSCs) could represent an elegant source for the generation of patient-specific cardiomyocytes needed for regenerative medicine, cardiovascular research, and pharmacological studies. However, the differentiation of adult MSC into a cardiac lineage is challenging compared to embryonic stem cells or induced pluripotent stem cells. Here we used non-integrative methods, including microRNA and mRNA, for cardiac reprogramming of adult MSC derived from bone marrow, dental follicle, and adipose tissue. We found that MSC derived from adipose tissue can partly be reprogrammed into the cardiac lineage by transient overexpression of GATA4, TBX5, MEF2C, and MESP1, while cells isolated from bone marrow, and dental follicle exhibit only weak reprogramming efficiency. qRT-PCR and transcriptomic analysis revealed activation of a cardiac-specific gene program and up-regulation of genes known to promote cardiac development. Although we did not observe the formation of fully mature cardiomyocytes, our data suggests that adult MSC have the capability to acquire a cardiac-like phenotype when treated with mRNA coding for transcription factors that regulate heart development. Yet, further optimization of the reprogramming process is mandatory to increase the reprogramming efficiency.