Detection of Apoptotic Cells in Vitro: A Practical Standardized Experimental Protocol Using Human Fibroblasts for Initial Laboratory Studies

This technical report presents a practical experimental protocol using human fibroblasts that is useful for initiating in situ methods for detection of apoptotic cells in vitro. The protocol details the growth of cells in multichamber wells, and includes a negative control, two positive controls, and untreated fibroblasts to be evaluated for apoptosis localization. Technical tips on handling of solutions, cell culture designs, and separation of slides by treatment are valuable steps for the laboratory beginning such studies. The methods proposed are both time and cost effective.     

In Vitro Comparison of Raypex 6 and Endopilot Using a Novel,Computer-Aided Measurement System,for Determining the Working Length

Introduction

Experimental evaluation of endometric devices usually relies on visual, subjective detection of the apical constriction to determine the accuracy of measurements. The aim of the present study was to analyze the accuracy of measurements of Raypex 6 and EndoPilot using a novel, objective image-analysis system.

Methods

Onehundred and twenty teeth were randomized and allocated to three groups: After coronal flaring, either Raypex 6 or EndoPilot were used to determine the endodontic working length during instrumentation using manual files (RPM and EPM group respectively). In addition, EndoPilot was used for continuous, automatic measurement during rotating instrumentation (EPA group). If the working length had been reached according to endometric results, the files were fixed in place. Tooth and file were then embedded and prepared for analysis. Subsequently, the distance between the tip of the file and the apical constriction (D_AC) or the apical foramen (D_AF) was calculated using trigonometric analysis and the position of the file relative to AC and AF was analyzed.

Results

Both inter- and intra-examiner-reliability of the trigonometric analysis were nearly perfect (ICC = 0.999, p<0.001). D_AC was not significantly different between groups (p>0.05, t-test). D_AF was significantly decreased when EPA had been used compared to EPM (p<0.05, Exact-test). EPA resulted in files being positioned beyond AF significantly more often than the other two methods (p<0.01).

Conclusions

All methods allowed reliable detection of AC. However, EPA significantly increased the risk of overpreparation. Objective, digital assessment based on image analysis was suitable to compare the accuracy of different endometric devices.     

A Novel In Vitro Method for Detecting Undifferentiated Human Pluripotent Stem Cells as Impurities in Cell Therapy Products Using a Highly Efficient Culture System

Innovative applications of cell therapy products (CTPs) derived from human pluripotent stem cells (hPSCs) in regenerative medicine are currently being developed. The presence of residual undifferentiated hPSCs in CTPs is a quality concern associated with tumorigencity. However, no simple in vitro method for direct detection of undifferentiated hPSCs that contaminate CTPs has been developed. Here, we show a novel approach for direct and sensitive detection of a trace amount of undifferentiated human induced pluripotent stem cells (hiPSCs) using a highly efficient amplification method in combination with laminin-521 and Essential 8 medium. Essential 8 medium better facilitated the growth of hiPSCs dissociated into single cells on laminin-521 than in mTeSR1 medium. hiPSCs cultured on laminin-521 in Essential 8 medium were maintained in an undifferentiated state and they maintained the ability to differentiate into various cell types. Essential 8 medium allowed robust hiPSC proliferation plated on laminin-521 at low cell density, whereas mTeSR1 did not enhance the cell growth. The highly efficient culture system using laminin-521 and Essential 8 medium detected hiPSCs spiked into primary human mesenchymal stem cells (hMSCs) or human neurons at the ratio of 0.001%–0.01% as formed colonies. Moreover, this assay method was demonstrated to detect residual undifferentiated hiPSCs in cell preparations during the process of hMSC differentiation from hiPSCs. These results indicate that our highly efficient amplification system using a combination of laminin-521 and Essential 8 medium is able to detect a trace amount of undifferentiated hPSCs contained as impurities in CTPs and would contribute to quality assessment of hPSC-derived CTPs during the manufacturing process.     

Basal Level of Autophagy Is Increased in Aging Human Skin Fibroblasts In Vitro,but Not in Old Skin

Intracellular autophagy (AP) is a stress response that is enhanced under conditions of limitation of amino acids, growth factors and other nutrients, and also when macromolecules become damaged, aggregated and fibrillated. Aging is generally accompanied by an increase in intracellular stress due to all the above factors. Therefore, we have compared the basal levels of AP in serially passaged human facial skin fibroblasts undergoing aging and replicative senescence in vitro, and ex vivo in the skin biopsies from the photo-protected and photo-exposed area of the arms of 20 healthy persons of young and old ages. Immunofluorescence microscopy, employing antibodies against a specific intracellular microtubule-associated protein-1 light chain-3 (LC3) as a well established marker of AP, showed a 5-fold increase in the basal level of LC3 in near senescent human skin fibroblasts. However, no such age-related increase in LC3 fluorescence and AP could be detected in full thickness skin sections from the biopsies obtained from 10 healthy young (age 25 to 30 yr) and 10 old (age 60 to 65 yr) donors. Furthermore, there was no difference in the basal level of LC3 in the skin sections from photo-protected and photo-exposed areas of the arm. Thus, in normal conditions, the aging phenotype of the skin cells in culture and in the body appears to be different in the case of AP.     

Assessment of Human Multi-Potent Hematopoietic Stem/Progenitor Cell Potential Using a Single In Vitro Screening System

Hematopoietic stem cells are responsible for the generation of the entire blood system through life. This characteristic relies on their ability to self renew and on their multi-potentiality. Thus quantification of the number of hematopoietic stem cells in a given cell population requires to show both properties in the studied cell populations. Although xenografts models that support human hematopoietic stem cells have been described, such in vivo experimental systems remain restrictive for high throughput screening purposes for example. In this work we developed a conditional tetracycline inducible system controlling the expression of the human NOTCH ligand Delta-like 1 in the murine stromal MS5 cells. We cultured hematopoietic immature cells enriched in progenitor/stem cells in contact with MS5 cells that conditionally express Delta-like 1, in conditions designed to generate multipotential lineage differentiation. We show that upon induction or repression of DL1 expression during co-culture, human immature CD34⁺CD38^−/low(CD45RA⁻CD90⁺) cells can express their B, T, NK, granulo/monocytic and erythroid potentials in a single well, and at the single cell level. We also document the interference of low NOTCH activation with human B and myelo/erythroid lymphoid differentiation. This system represents a novel tool to precisely quantify human hematopoietic immature cells with both lymphoid and myeloid potentials.     

Detection of DNA Damage in Cultured Human Fibroblasts Induced by Methyl Linoleate Hydroperoxide

To establish a strategy to generate N-acylated proteins modified with fatty acids having a specific chain length, tGelsolin-streptag, an epitope-tagged model protein having an N-myristoylation motif, was synthesized using an insect cell-free protein synthesis system in the presence of acyl-CoA with various fatty acid chain lengths. It was found that the fatty acid species attached to the N-termini fully depended on the acyl-CoA species added to the reaction mixture. N-Acylated proteins with fatty acid chain lengths of 8, 10, 12, and 14 were generated successfully.     

In Vitro Selection of a Peptide Inhibitor of Human IL-6 Using mRNA Display

Interleukin-6 (IL-6) plays a crucial role in malignant diseases, such as rheumatoid arthritis, Castleman disease, and multiple myeloma, and as such, is an attractive therapeutic target. Here, the authors isolated a novel IL-6 inhibitor peptide by in vitro selection using mRNA display. The authors first used a random-primed human cDNA library to isolate IL-6-binding peptides. After four rounds of selection, a 19-amino acid peptide named CA11 was selected and confirmed to specifically interact with IL-6. The authors then performed an alanine scan analysis of CA11 and determined the amino acid residues necessary to interact with IL-6. Next, the authors constructed a CA11-based partially randomized library and after ten more rounds of selection, isolated several groups of peptides. The most frequently occurring sequence, RA07, bound to IL-6 with 3 to 4-fold higher affinity than CA11. Furthermore, RA07 inhibited IL-6-dependent KT-3 cell proliferation in a dose-dependent manner. ELISAs revealed that RA07 could not inhibit IL-6 from binding to the IL-6 receptor (IL-6R), but could inhibit the IL-6/IL-6 complex binding to gp130.     

Prediction of Chemical Respiratory Sensitizers Using GARD,a Novel In Vitro Assay Based on a Genomic Biomarker Signature

Background

Repeated exposure to certain low molecular weight (LMW) chemical compounds may result in development of allergic reactions in the skin or in the respiratory tract. In most cases, a certain LMW compound selectively sensitize the skin, giving rise to allergic contact dermatitis (ACD), or the respiratory tract, giving rise to occupational asthma (OA). To limit occurrence of allergic diseases, efforts are currently being made to develop predictive assays that accurately identify chemicals capable of inducing such reactions. However, while a few promising methods for prediction of skin sensitization have been described, to date no validated method, in vitro or in vivo, exists that is able to accurately classify chemicals as respiratory sensitizers.

Results

Recently, we presented the in vitro based Genomic Allergen Rapid Detection (GARD) assay as a novel testing strategy for classification of skin sensitizing chemicals based on measurement of a genomic biomarker signature. We have expanded the applicability domain of the GARD assay to classify also respiratory sensitizers by identifying a separate biomarker signature containing 389 differentially regulated genes for respiratory sensitizers in comparison to non-respiratory sensitizers. By using an independent data set in combination with supervised machine learning, we validated the assay, showing that the identified genomic biomarker is able to accurately classify respiratory sensitizers.

Conclusions

We have identified a genomic biomarker signature for classification of respiratory sensitizers. Combining this newly identified biomarker signature with our previously identified biomarker signature for classification of skin sensitizers, we have developed a novel in vitro testing strategy with a potent ability to predict both skin and respiratory sensitization in the same sample.     

Regulation of Collagen Synthesis in Human Dermal Fibroblasts in Contracted Collagen Gels by Ascorbic Acid, Growth Factors, and Inhibitors of Lipid Peroxidation

Ascorbic acid has been shown to stimulate collagen synthesis in monolayer cultures of human dermal fibroblasts. In the present studies, we examined whether the presence of a collagen matrix influences this response of dermal fibroblasts to ascorbic acid. Fibroblasts and collagen were mixed and allowed to gel and contract for 6 days to form a matrix prior to determining the concentration and time dependence for ascorbic acid to affect collagen synthesis by fibroblasts within the matrix. Collagen synthesis was stimulated at levels at or above 10 μM ascorbic acid and was maximal after 2 days of treatment. This concentration and time dependence is similar to that of cells grown in monolayer cultures. The effects of transforming growth factor-β (TGF-β) and fibroblast growth factor (FGF) were also examined in this model. TGF-β increased and FGF inhibited collagen synthesis in the gels, as has been shown for cells in monolayer cultures. The effects of potential inhibitors of lipid peroxidation induced by ascorbic acid were also examined in these matrices and compared to previous results obtained in monolayer cultures. Propyl gallate, cobalt chloride, α,α-dipyridyl, and α-tocopherol inhibited the ascorbic acid-mediated stimulation of collagen synthesis while mannitol had no effect. Natural retinoids inhibited total protein synthesis without the specific effect on collagen synthesis that was seen in monolayer cultures. These results indicate that ascorbic acid stimulates collagen synthesis in fibroblasts grown in a collagen matrix in a manner similar to that found in monolayer cultures. In contracting collagen gels, however, the magnitude of the effect is less and retinoids do not specifically inhibit collagen synthesis.     

Coupling of Human DNA Excision Repair and the DNA Damage Checkpoint in a Defined in Vitro System

DNA repair and DNA damage checkpoints work in concert to help maintain genomic integrity. In vivo data suggest that these two global responses to DNA damage are coupled. It has been proposed that the canonical 30 nucleotide single-stranded DNA gap generated by nucleotide excision repair is the signal that activates the ATR-mediated DNA damage checkpoint response and that the signal is enhanced by gap enlargement by EXO1 (exonuclease 1) 5′ to 3′ exonuclease activity. Here we have used purified core nucleotide excision repair factors (RPA, XPA, XPC, TFIIH, XPG, and XPF-ERCC1), core DNA damage checkpoint proteins (ATR-ATRIP, TopBP1, RPA), and DNA damaged by a UV-mimetic agent to analyze the basic steps of DNA damage checkpoint response in a biochemically defined system. We find that checkpoint signaling as measured by phosphorylation of target proteins by the ATR kinase requires enlargement of the excision gap generated by the excision repair system by the 5′ to 3′ exonuclease activity of EXO1. We conclude that, in addition to damaged DNA, RPA, XPA, XPC, TFIIH, XPG, XPF-ERCC1, ATR-ATRIP, TopBP1, and EXO1 constitute the minimum essential set of factors for ATR-mediated DNA damage checkpoint response.     

Establishment of a Novel In Vitro Test Setup for Electric and Magnetic Stimulation of Human Osteoblasts

When large defects occur, bone regeneration can be supported by bone grafting and biophysical stimuli like electric and magnetic stimulation (EMS). Clinically established EMS modes are external coils and surgical implants like an electroinductive screw system, which combines a magnetic and electric field, e.g., for the treatment of avascular bone necrosis or pseudarthrosis. For optimization of this implant system, an in vitro test setup was designed to investigate effects of EMS on human osteoblasts on different 3D scaffolds (based on calcium phosphate and collagen). Prior to the cell experiments, numerical simulations of the setup, as well as experimental validation, via measurements of the electric parameters induced by EMS were conducted. Human osteoblasts (3 × 10⁵ cells) were seeded onto the scaffolds and cultivated. After 24 h, screw implants (Stryker ASNIS III s-series) were centered in the scaffolds, and EMS was applied (3 × 45 min per day at 20 Hz) for 3 days. Cell viability and collagen type 1 (Col1) synthesis were determined subsequently. Numerical simulation and validation showed an adequate distribution of the electric field within the scaffolds. Experimental measurements of the electric potential revealed only minimal deviation from the simulation. Cell response to stimulation varied with scaffold material and mode of stimulation. EMS-stimulated cells exhibited a significant decrease of metabolic activity in particular on collagen scaffolds. In contrast, the Col1/metabolic activity ratio was significantly increased on collagen and non-sintered calcium phosphate scaffolds after 3 days. Exclusive magnetic stimulation showed similar but nonsignificant tendencies in metabolic activity and Col1 synthesis. The cell tests demonstrate that the new test setup is a valuable tool for in vitro testing and parameter optimization of the clinically used electroinductive screw system. It combines magnetic and electric stimulation, allowing in vitro investigations of its influence on human osteoblasts.     

In Vitro Antimicrobial Activity and Human Pharmacology of Cephalexin, a New Orally Absorbed Cephalosporin C Antibiotic

Concentrations of cephalexin (an orally absorbed derivative of cephalosporin C) in serum and urine were determined in normal volunteers and patients. The in vitro antibacterial activity was also studied. All strains of group A β-hemolytic streptococci and Diplococcus pneumoniae were inhibited by 3.1 μg/ml. Of the Staphylococcus aureus strains, 88% were inhibited by 6.3 μg/ml, and 12.5 μg/ml was inhibitory for all S. aureus, 80% of Escherichia coli, 72% of Klebsiella-Aerobacter, and 56% of Proteus mirabilis strains. About 90 to 96% of E. coli, Klebsiella Aerobacter, and P. mirabilis strains were inhibited by 25 μg of cephalexin per ml. Pseudomonas and indole-positive Proteus strains proved to be quite resistant to cephalexin. Cephalexin was well absorbed after oral administration. A peak serum concentration of cephalexin of at least 5 μg/ml was achieved in each volunteer with 250 and 500-mg doses. A mean peak serum concentration of 7.7 μg/ml was achieved with 250-mg doses; 12.3μg/ml was achieved with 500-mg doses of antibiotic. Food did not interfere with absorption. Probenecid enhanced both the peak serum concentration and the duration of antibiotic activity in the serum. Over 90% of the administered dose was excreted in the urine within 6 hr. The mean peak serum concentration of cephalexin after an oral dose of 500 mg was adequate to inhibit all group A streptococci, D. pneumoniae, and S. aureus, 85% of E. coli, and about 40 to 75% of Klebsiella-Aerobacter and P. mirabilis strains. Levels of cephalexin in urine were adequate to inhibit over 90% of E. coli, and P. mirabilis and 80 to 96% of Klebsiella-Aerobacter strains.     

Systematic Analysis of In Vitro Cell Rolling Using a Multi-well Plate Microfluidic System

A major challenge for cell-based therapy is the inability to systemically target a large quantity of viable cells with high efficiency to tissues of interest following intravenous or intraarterial infusion. Consequently, increasing cell homing is currently studied as a strategy to improve cell therapy. Cell rolling on the vascular endothelium is an important step in the process of cell homing and can be probed in-vitro using a parallel plate flow chamber (PPFC). However, this is an extremely tedious, low throughput assay, with poorly controlled flow conditions. Instead, we used a multi-well plate microfluidic system that enables study of cellular rolling properties in a higher throughput under precisely controlled, physiologically relevant shear flow^1,2. In this paper, we show how the rolling properties of HL-60 (human promyelocytic leukemia) cells on P- and E-selectin-coated surfaces as well as on cell monolayer-coated surfaces can be readily examined. To better simulate inflammatory conditions, the microfluidic channel surface was coated with endothelial cells (ECs), which were then activated with tumor necrosis factor-α (TNF-α), significantly increasing interactions with HL-60 cells under dynamic conditions. The enhanced throughput and integrated multi-parameter software analysis platform, that permits rapid analysis of parameters such as rolling velocities and rolling path, are important advantages for assessing cell rolling properties in-vitro. Allowing rapid and accurate analysis of engineering approaches designed to impact cell rolling and homing, this platform may help advance exogenous cell-based therapy.     

In Vitro and In Vivo Studies on Chitosan Beads of Losartan Duolite AP143 Complex, Optimized by Using Statistical Experimental Design

The aim of the present research work was to develop release modulated beads of losartan potassium complexed with anion exchange resin, Duolite AP143 (cholestyramine). Chitosan was selected as a hydrophilic polymer for the formation of beads which could sustain the release of the drug up to 12 h, along with drug resin complex (DRC). Chitosan beads were prepared using an in-liquid curing method by ionotropic cross-linking or interpolymer linkage with sodium tripolyphosphate (TPP). The formulation of the beads was optimized for entrapment efficiency and drug release using 3² full factorial design. The independent variables selected were DRC/chitosan and percent of TPP. The optimization model was validated for its performance characteristics. Studies revealed that as the concentration of chitosan and TPP was increased, entrapment efficiency and the drug release were found to increase and decrease, respectively. The swelling capacity of chitosan–TPP beads decreased with increasing concentration of TPP. The effect of chitosan concentration and percentage of TPP solution used for cross-linking on entrapment efficiency and drug release rate was extensively investigated. Optimized beads were subjected to in vivo studies in Wistar albino rats to determine the mean arterial blood pressure and compared with marketed formulation. The pharmacodynamic study demonstrates steady blood pressure control for optimized formulation as compared to fluctuated blood pressure for the marketed formulation.     

Induction of G1 Cell Cycle Arrest in Human Glioma Cells by Salinomycin Through Triggering ROS-Mediated DNA Damage In Vitro and In Vivo

Chemotherapy has always been one of the most effective ways in combating human glioma. However, the high metastatic potential and resistance toward standard chemotherapy severely hindered the chemotherapy outcomes. Hence, searching effective chemotherapy drugs and clarifying its mechanism are of great significance. Salinomycin an antibiotic shows novel anticancer potential against several human tumors, including human glioma, but its mechanism against human glioma cells has not been fully elucidated. In the present study, we demonstrated that salinomycin treatment time- and dose-dependently inhibited U251 and U87 cells growth. Mechanically, salinomycin-induced cell growth inhibition against human glioma was mainly achieved by induction of G1-phase arrest via triggering reactive oxide species (ROS)-mediated DNA damage, as convinced by the activation of histone, p53, p21 and p27. Furthermore, inhibition of ROS accumulation effectively attenuated salinomycin-induced DNA damage and G1 cell cycle arrest, and eventually reversed salinomycin-induced cytotoxicity. Importantly, salinomycin treatment also significantly inhibited the U251 tumor xenograft growth in vivo through triggering DNA damage-mediated cell cycle arrest with involvement of inhibiting cell proliferation and angiogenesis. The results above validated the potential of salinomycin-based chemotherapy against human glioma.     

Anticancer Activity of MPT0E028, a Novel Potent Histone Deacetylase Inhibitor, in Human Colorectal Cancer HCT116 Cells In Vitro and In Vivo

Recently, histone deacetylase (HDAC) inhibitors have emerged as a promising class of drugs for treatment of cancers, especially subcutaneous T-cell lymphoma. In this study, we demonstrated that MPT0E028, a novel N-hydroxyacrylamide-derived HDAC inhibitor, inhibited human colorectal cancer HCT116 cell growth in vitro and in vivo. The results of NCI-60 screening showed that MPT0E028 inhibited proliferation in both solid and hematological tumor cell lines at micromolar concentrations, and was especially potent in HCT116 cells. MPT0E028 had a stronger apoptotic activity and inhibited HDACs activity more potently than SAHA, the first therapeutic HDAC inhibitor proved by FDA. In vivo murine model, the growth of HCT116 tumor xenograft was delayed and inhibited after treatment with MPT0E028 in a dose-dependent manner. Based on in vivo study, MPT0E028 showed stronger anti-cancer efficacy than SAHA. No significant body weight difference or other adverse effects were observed in both MPT0E028-and SAHA-treated groups. Taken together, our results demonstrate that MPT0E028 has several properties and is potential as a promising anti-cancer therapeutic drug.