Isolation of Human Progenitor Epidermal Cells on Collagen Type IV and Analysis of Their Markers with Flow Cytometry and PCR Methods.

Epidermal stem cells, located in the skin, together with keratinocytes are transplanted in regenerative therapies, e.g., for the treatment of burns or other wounds. Here, we describe the protocol of their enzymatic isolation from human skin. It includes separation of the epidermis form the dermis by incubation with dispase followed by cell isolation for epidermis by digestion with trypsin. Cell isolated with this method can be seeded on collagen IV-coated dishes. The methods of analysis of epidermal stem cells markers (e.g., CD71, CD29) with flow cytometry and RT-PCR are also included.

Broadband Electrical Spectroscopy to Distinguish Single-Cell Ca2+ Changes Due to Ionomycin Treatment in a Skeletal Muscle Cell Line.

Many skeletal muscle diseases such as muscular dystrophy, myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), and sarcopenia share the dysregulation of calcium (Ca2+) as a key mechanism of disease at a cellular level. Cytosolic concentrations of Ca2+ can signal dysregulation in organelles including the mitochondria, nucleus, and sarcoplasmic reticulum in skeletal muscle. In this work, a treatment is applied to mimic the Ca2+ increase associated with these atrophy-related disease states, and broadband impedance measurements are taken for single cells with and without this treatment using a microfluidic device. The resulting impedance measurements are fitted using a single-shell circuit simulation to show calculated electrical dielectric property contributions based on these Ca2+ changes. From this, similar distributions were seen in the Ca2+ from fluorescence measurements and the distribution of the S-parameter at a single frequency, identifying Ca2+ as the main contributor to the electrical differences being identified. Extracted dielectric parameters also showed different distribution patterns between the untreated and ionomycin-treated groups; however, the overall electrical parameters suggest the impact of Ca2+-induced changes at a wider range of frequencies.

Microfluidic Impedance Cytometry for Single-Cell Particulate Inorganic Carbon:Particulate Organic Carbon Measurements of Calcifying Algae.

Calcifying algae, like coccolithophores, greatly contribute to the oceanic carbon cycle and are therefore of particular interest for ocean carbon models. They play a key role in two processes that are important for the effective CO2 flux: The organic carbon pump (photosynthesis) and the inorganic carbon pump (calcification). The relative contribution of calcification and photosynthesis can be measured in algae by the amount of particulate inorganic carbon (PIC) and particulate organic carbon (POC). A microfluidic impedance cytometer is presented, enabling non-invasive and high-throughput assessment of the calcification state of single coccolithophore cells. Gradual modification of the exoskeleton by acidification results in a strong linear fit (R 2 = 0.98) between the average electrical phase and the PIC:POC ratio of the coccolithophore Emiliania huxleyi 920/9. The effect of different CO2 treatments on the PIC:POC ratio, however, is inconclusive, indicating that there is no strong effect observed for this particular strain. Lower PIC:POC ratios in cultures that grew to higher cell densities are found, which are also recorded with the impedance-based PIC:POC sensor. The development of this new quantification tool for small volumes paves the way for high-throughput analysis while applying multi-variable environmental stressors to support projections of the future marine carbon cycle.

Stem cell sources and characterization in the development of cell-based products for treating retinal disease: An NEI Town Hall report.

National Eye Institute recently issued a new Strategic Plan outlining priority research areas for the next 5 years. Starting cell source for deriving stem cell lines is as an area with gaps and opportunities for making progress in regenerative medicine, a key area of emphasis within the NEI Strategic Plan. There is a critical need to understand how starting cell source affects the cell therapy product and what specific manufacturing capabilities and quality control standards are required for autologous vs allogeneic stem cell sources. With the goal of addressing some of these questions, in discussion with the community-at-large, NEI hosted a Town Hall at the Association for Research in Vision and Ophthalmology annual meeting in May 2022. This session leveraged recent clinical advances in autologous and allogeneic RPE replacement strategies to develop guidance for upcoming cell therapies for photoreceptors, retinal ganglion cells, and other ocular cell types. Our focus on stem cell-based therapies for RPE underscores the relatively advanced stage of RPE cell therapies to patients with several ongoing clinical trials. Thus, this workshop encouraged lessons learned from the RPE field to help accelerate progress in developing stem cell-based therapies in other ocular tissues. This report provides a synthesis of the key points discussed at the Town Hall and highlights needs and opportunities in ocular regenerative medicine.

Process Technologies of Cyanobacteria.

Although the handling and exploitation of cyanobacteria is associated with some challenges, these phototrophic bacteria offer great opportunities for innovative biotechnological processes. This chapter covers versatile aspects of working with cyanobacteria, starting with up-to-date in silico and in vitro screening methods for bioactive substances. Subsequently, common conservation techniques and vitality/viability estimation methods are compared and supplemented by own data regarding the non-invasive vitality evaluation via pulse amplitude modulated fluorometry. Moreover, novel findings about the influence the state of the pre-cultures have on main cultures are presented. The following sub-chapters deal with different photobioreactor-designs, with special regard to biofilm photobioreactors, as well as with heterotrophic and mixotrophic cultivation modes. The latter topic provides information from literature on successfully enhanced cyanobacterial production processes, augmented by own data.

Human lung cell models to study aerosol delivery - considerations for model design and development.

Human lung tissue models range from simple monolayer cultures to more advanced three-dimensional co-cultures. Each model system can address the interactions of different types of aerosols and the choice of the model and the mode of aerosol exposure depends on the relevant scenario, such as adverse outcomes and endpoints of interest. This review focuses on the functional, as well as structural, aspects of lung tissue from the upper airway to the distal alveolar compartments as this information is relevant for the design of a model as well as how the aerosol properties determine the interfacial properties with the respiratory wall. The most important aspects on how to design lung models are summarized with a focus on (i) choice of appropriate scaffold, (ii) selection of cell types for healthy and diseased lung models, (iii) use of culture condition and assembly, (iv) aerosol exposure methods, and (v) endpoints and verification process. Finally, remaining challenges and future directions in this field are discussed.

Medium supplementation can influence the human ovarian cells in vitro.

BACKGROUND: Cells are an essential part of the triple principles of tissue engineering and a crucial component of the engineered ovary as they can induce angiogenesis, synthesize extracellular matrix and influence follicle development. Here, we hypothesize that by changing the medium supplementation, we can obtain different cell populations isolated from the human ovary to use in the engineered ovary. To this end, we have in vitro cultured cells isolated from the menopausal ovarian cortex using different additives: KnockOut serum replacement (KO), fetal bovine serum (FBS), human serum albumin (HSA), and platelet lysate (PL). RESULTS: Our results showed that most cells soon after isolation (pre-culture, control) and cells in KO and FBS groups were CD31- CD34- (D0: vs. CD31-CD34+, CD31 + CD34+, and CD31 + CD34- p < 0.0001; KO: vs. CD31-CD34+, CD31 + CD34+, and CD31 + CD34- p < 0.0001; FBS: vs. CD31-CD34+ and CD31 + CD34+ p < 0.001, and vs. CD31 + CD34- p < 0.01). Moreover, a deeper analysis of the CD31-CD34- population demonstrated a significant augmentation (more than 86%) of the CD73+ and CD90+ cells (possibly fibroblasts, mesenchymal stem cells, or pericytes) in KO- and FBS-based media compared to the control (around 16%; p < 0.001). Still, in the CD31-CD34- population, we found a higher proportion (60%) of CD90+ and PDPN+ cells (fibroblast-like cells) compared to the control (around 7%; vs PL and KO p < 0.01 and vs FBS p < 0.001). Additionally, around 70% of cells in KO- and FBS-based media were positive for CD105 and CD146, which may indicate an increase in the number of pericytes in these media compared to a low percentage (4%) in the control group (vs KO and FBS p < 0.001). On the other hand, we remarked a significant decrease of CD31- CD34+ cells after in vitro culture using all different medium additives (HSA vs D0 p < 0.001, PL, KO, and FBS vs D0 P < 0.01). We also observed a significant increase in epithelial cells (CD326+) when the medium was supplemented with KO (vs D0 p < 0.05). Interestingly, HSA and PL showed more lymphatic endothelial cells compared to other groups (CD31 + CD34+: HSA and PL vs KO and FBS p < 0.05; CD31 + CD34 + CD90 + PDPN+: HSA and PL vs D0 p < 0.01). CONCLUSION: Our results demonstrate that medium additives can influence the cell populations, which serve as building blocks for the engineered tissue. Therefore, according to the final application, different media can be used in vitro to favor different cell types, which will be incorporated into a functional matrix.

Performance Analysis of a CSFH-Based Microgripper: Analytical Modeling and Simulation.

Microgrippers are promising tools for micro-manipulation and characterization of cells. In this paper, a biocompatible electro-thermally actuated microgripper with rotary capacitive position sensor is presented. To overcome the limited displacement possibilities usually provided by electrothermal actuators and to achieve the desired tweezers output displacement, conjugate surface flexure hinges (CSFH) are adopted. The microgripper herein reported can in principle manipulate biological samples in the size range between 15 and 120 µm. A kinematics modeling approach based on the pseudo-rigid-body-method (PRBM) is applied to describe the microgripper's working mechanism, and analytical modeling, based on finite elements method (FEM), is used to optimize the electrothermal actuator design and the heat dissipation mechanism. Finally, FEM-based simulations are carried out to verify the microgripper, the electrothermal actuator and heat dissipation mechanism performance, and to assess the validity of the analytical modeling.

Life at the periphery: what makes CHO cells survival talents.

The production of biopharmaceuticals relies on robust cell systems that can produce recombinant proteins at high levels and grow and survive in the stressful bioprocess environment. Chinese hamster ovary cells (CHO) as the main production hosts offer a variety of advantages including robust growth and survival in a bioprocess environment. Cell surface proteins are of special interest for the understanding of how CHO cells react to their environment while maintaining growth and survival phenotypes, since they enable cellular reactions to external stimuli and potentially initiate signaling pathways. To provide deeper insight into functions of this special cell surface sub-proteome, pathway enrichment analysis of the determined CHO surfaceome was conducted. Enrichment of growth/ survival-pathways such as the phosphoinositide-3-kinase (PI3K)-protein kinase B (AKT), mitogen-activated protein kinase (MAPK), Janus kinase/signal transducers and activators of transcription (JAK-STAT), and RAP1 pathways were observed, offering novel insights into how cell surface receptors and ligand-mediated signaling enable the cells to grow and survive in a bioprocess environment. When supplementing surfaceome data with RNA expression data, several growth/survival receptors were shown to be co-expressed with their respective ligands and thus suggesting self-induction mechanisms, while other receptors or ligands were not detectable. As data about the presence of surface receptors and their associated expressed ligands may serve as base for future studies, further pathway characterization will enable the implementation of optimization strategies to further enhance cellular growth and survival behavior. KEY POINTS: • PI3K/AKT, MAPK, JAK-STAT, and RAP1 pathway receptors are enriched on the CHO cell surface and downstream pathways present on mRNA level. • Detected pathways indicate strong CHO survival and growth phenotypes. • Potential self-induction of surface receptors and respective ligands.

Optimisation of processing methods to improve success in the derivation of human multipotent mesenchymal stromal cells from cryopreserved umbilical cord tissue fragments.

Wharton's Jelly (WJ)-derived Mesenchymal Stromal Cells (MSC) are currently in the spotlight for the development of innovative MSC-based therapies due to their ease of sourcing, high proliferation capacity and improved immunopotency over MSC from other tissue sources. However, the short time window for derivation from donated fresh umbilical cord (UC) tissue fragments does not allow to consider biological features of the donor beyond serological safety testing. This limits the scope of MSC banking to rapid, prospective derivation of MSC, WJ lines without considering biological and genetic characteristics of the donor that may influence their suitability for clinical use (e.g. HLA type, inherited gene variants). In the present study, we describe a simple, efficient and reproducible approach for the cryopreservation of UC tissue fragments, compatible with established workflows in existing public frameworks for cord blood and tissue collection while guaranteeing pharmaceutical grade of starting materials for further processing under GMP standards. Herein we demonstrated the feasibility of time and cost-saving methods for cryopreservation of unprocessed UC tissue fragments directly at reception of the donated tissues using 10% Me2SO-based cryosolution and a commercial clinical-grade defined cryopreservation medium (Cryostor®), showing the preservation of all Critical Quality Attributes in terms of identity, potency and kinetic parameters. In summary, our study provides evidence that cryopreservation of large unprocessed UC tissue fragments (5-13.5 cm) supports subsequent progenitor cell isolation and derivation of MSC,WJ, preserving their viability, identity, proliferation rates and potency.

Techniques for Profiling the Cellular Immune Response and Their Implications for Interventional Oncology.

In recent years there has been increased interest in using the immune contexture of the primary tumors to predict the patient's prognosis. The tumor microenvironment of patients with cancers consists of different types of lymphocytes, tumor-infiltrating leukocytes, dendritic cells, and others. Different technologies can be used for the evaluation of the tumor microenvironment, all of which require a tissue or cell sample. Image-guided tissue sampling is a cornerstone in the diagnosis, stratification, and longitudinal evaluation of therapeutic efficacy for cancer patients receiving immunotherapies. Therefore, interventional radiologists (IRs) play an essential role in the evaluation of patients treated with systemically administered immunotherapies. This review provides a detailed description of different technologies used for immune assessment and analysis of the data collected from the use of these technologies. The detailed approach provided herein is intended to provide the reader with the knowledge necessary to not only interpret studies containing such data but also design and apply these tools for clinical practice and future research studies.

Synchronized, Spontaneous, and Oscillatory Detachment of Eukaryotic Cells: A New Tool for Cell Characterization and Identification.

Despite the importance of cell characterization and identification for diagnostic and therapeutic applications, developing fast and label-free methods without (bio)-chemical markers or surface-engineered receptors remains challenging. Here, we exploit the natural cellular response to mild thermal stimuli and propose a label- and receptor-free method for fast and facile cell characterization. Cell suspensions in a dedicated sensor are exposed to a temperature gradient, which stimulates synchronized and spontaneous cell-detachment with sharply defined time-patterns, a phenomenon unknown from literature. These patterns depend on metabolic activity (controlled through temperature, nutrients, and drugs) and provide a library of cell-type-specific indicators, allowing to distinguish several yeast strains as well as cancer cells. Under specific conditions, synchronized glycolytic-type oscillations are observed during detachment of mammalian and yeast-cell ensembles, providing additional cell-specific signatures. These findings suggest potential applications for cell viability analysis and for assessing the collective response of cancer cells to drugs.

A Microfluidic Dielectric Spectroscopy System for Characterization of Biological Cells in Physiological Media.

Dielectric spectroscopy (DS) is a promising cell screening method that can be used for diagnostic and drug discovery purposes. The primary challenge of using DS in physiological buffers is the electrode polarization (EP) that overwhelms the impedance signal within a large frequency range. These effects further amplify with the miniaturization of the measurement electrodes. In this study, we present a microfluidic system and the associated equivalent circuit models for real-time measurements of cell membrane capacitance and cytoplasm resistance in physiological buffers with 10 s increments. The current device captures several hundreds of biological cells in individual microwells through gravitational settling and measures the system's impedance using microelectrodes covered with dendritic gold nanostructures. Using PC-3 cells (a highly metastatic prostate cancer cell line) suspended in cell growth media (CGM), we demonstrate stable measurements of cell membrane capacitance and cytoplasm resistance in the device for over 15 min. We also describe a consistent application of the equivalent circuit model, starting from the reference measurements used to determine the system parameters. The circuit model is tested using devices with varying dimensions, and the obtained cell parameters between different devices are nearly identical. Further analyses of the impedance data have shown that accurate cell membrane capacitance and cytoplasm resistance can be extracted using a limited number of measurements in the 5 MHz to 10 MHz range. This will potentially reduce the timescale required for real-time DS measurements below 1 s. Overall, the new microfluidic device can be used for the dielectric characterization of biological cells in physiological buffers for various cell screening applications.

On-Chip Optical Nano-Tweezers for Culture-Less Fast Bacterial Viability Assessment.

Because of antibiotics misuse, the dramatic growth of antibioresistance threatens public health. Tests are indeed culture-based, and require therefore one to two days. This long time-to-result implies the use of large-spectrum antibiotherapies as a first step, in absence of pathogen characterization. Here, a breakthrough approach for a culture-less fast assessment of bacterial response to stress is proposed. It is based on non-destructive on-chip optical tweezing. A laser loads an optical nanobeam cavity whose evanescent part of the resonant field acts as a nano-tweezer for bacteria surrounding the cavity. Once optically trapped, the bacterium-nanobeam cavity interaction induces a shift of the resonance driven by the bacterial cell wall optical index. The analysis of the wavelength shift yields an assessment of viability upon stress at the single-cell scale. As a proof of concept, bacteria are stressed by incursion, before optical trapping, at different temperatures (45, 51, and 70 °C). Optical index changes correlate with the degree of thermal stress allowing to sort viable and dead bacteria. With this disruptive diagnosis method, bacterial viability upon stress is probed much faster (typically less than 4 h) than with conventional culture-based enumeration methods (24 h).

Comparative Evaluation of Mouse Bone Marrow Mesenchymal Stromal Cells Characteristics Cultured in Two Different Supplemented Media

@#Introduction: Preclinical studies on mesenchymal stromal cells (MSC) have allowed the cells to be considered as a promising candidate for cellular therapy. In recent years, conflicting data have been reported regarding various aspects of their characteristics, development and differentiation potential, which may be due to arrange of factors. Among the factors worth investigating is the culture medium formulation. Methods: Here we have made a comparative characterization of mouse bone marrow mesenchymal stromal cells (mBM-MSC) that were cultured using two common supplements, MesenCult™ Stimulatory Supplement (MSS) and fetal bovine serum (FBS), under the same experimental conditions at different passages. Clonogenic potential, cumulative population doubling level (CPDL), population doubling time (PDT), immunophenotyping, differentiation, immunosuppression potentials and chromosome analysis of early and late passages mBM-MSC were assessed. Results: Our findings showed that the CPDL, immunophenotype and immunosuppression potential of mBM-MSC were similar. However, variations were seen in their clonogenicity, population doubling time and differentiation efficacy whereby all of these were enhanced in DMSS. These observations suggest that their genetic make-up may be affected by both supplements upon prolonged culture. Interestingly, this conjecture was supported when chromosomal analysis revealed genetic instability of mBM-MSCs cultured in both supplements. Conclusion: In conclusion, culture medium formulation was shown to cause variations and spontaneous transformation in mBM-MSCs raising concerns on the usage of late passages mBMMSCs in fundamental and preclinical downstream experiments.

Intra-Individual Variability of Human Dental Pulp Stem Cell Features Isolated from the Same Donor.

It is primarily important to define the standard features and factors that affect dental pulp stem cells (DPSCs) for their broader use in tissue engineering. This study aimed to verify whether DPSCs isolated from various teeth extracted from the same donor exhibit intra-individual variability and what the consequences are for their differentiation potential. The heterogeneity determination was based on studying the proliferative capacity, viability, expression of phenotypic markers, and relative length of telomere chromosomes. The study included 14 teeth (6 molars and 8 premolars) from six different individuals ages 12 to 16. We did not observe any significant intra-individual variability in DPSC size, proliferation rate, viability, or relative telomere length change within lineages isolated from different teeth but the same donor. The minor non-significant variances in phenotype were probably mainly because DPSC cell lines comprised heterogeneous groups of undifferentiated cells independent of the donor. The other variances were seen in DPSC lineages isolated from the same donor, but the teeth were in different stages of root development. We also did not observe any changes in the ability of cells to differentiate into mature cell lines-chondrocytes, osteocytes, and adipocytes. This study is the first to analyze the heterogeneity of DPSC dependent on a donor.

Novel, Fully Characterised Bovine Taste Bud Cells of Fungiform Papillae.

Current understanding of functional characteristics and biochemical pathways in taste bud cells have been hindered due the lack of long-term cultured cells. To address this, we developed a holistic approach to fully characterise long term cultured bovine taste bud cells (BTBCs). Initially, cultured BTBCs were characterised using RT-PCR gene expression profiling, immunocytochemistry, flowcytometry and calcium imaging, that confirmed the cells were mature TBCs that express taste receptor genes, taste specific protein markers and capable of responding to taste stimuli, i.e., denatonium (2 mM) and quinine (462.30 µM). Gene expression analysis of forty-two genes implicated in taste transduction pathway (map04742) using custom-made RT-qPCR array revealed high and low expressed genes in BTBCs. Preliminary datamining and bioinformatics demonstrated that the bovine α-gustducin, gustatory G-protein, have higher sequence similarity to the human orthologue compared to rodents. Therefore, results from this work will replace animal experimentation and provide surrogate cell-based throughput system to study human taste transduction.

Cellular Heterogeneity of Mesenchymal Stem/Stromal Cells in the Bone Marrow.

Mesenchymal stem/stromal cells (MSCs) are present in various body tissues and help in maintaining homeostasis. The stemness of MSCs has been evaluated in vitro. In addition, analyses of cell surface antigens and gene expression patterns have shown that MSCs comprise a heterogeneous population, and the diverse and complex nature of MSCs makes it difficult to identify the specific roles in diseases. There is a lack of understanding regarding the classification of MSC properties. In this review, we explore the characteristics of heterogeneous MSC populations based on their markers and gene expression profiles. We integrated the contents of previously reported single-cell analysis data to better understand the properties of mesenchymal cell populations. In addition, the cell populations involved in the development of myeloproliferative neoplasms (MPNs) are outlined. Owing to the diversity of terms used to describe MSCs, we used the text mining technology to extract topics from MSC research articles. Recent advances in technology could improve our understanding of the diversity of MSCs and help us evaluate cell populations.

Characterization and Separation of Live and Dead Yeast Cells Using CMOS-Based DEP Microfluidics.

This study aims at developing a miniaturized CMOS integrated silicon-based microfluidic system, compatible with a standard CMOS process, to enable the characterization, and separation of live and dead yeast cells (as model bio-particle organisms) in a cell mixture using the DEP technique. DEP offers excellent benefits in terms of cost, operational power, and especially easy electrode integration with the CMOS architecture, and requiring label-free sample preparation. This can increase the likeliness of using DEP in practical settings. In this work the DEP force was generated using an interdigitated electrode arrays (IDEs) placed on the bottom of a CMOS-based silicon microfluidic channel. This system was primarily used for the immobilization of yeast cells using DEP. This study validated the system for cell separation applications based on the distinct responses of live and dead cells and their surrounding media. The findings confirmed the device's capability for efficient, rapid and selective cell separation. The viability of this CMOS embedded microfluidic for dielectrophoretic cell manipulation applications and compatibility of the dielectrophoretic structure with CMOS production line and electronics, enabling its future commercially mass production.