Clinostat 3D Cell Culture: Protocols for the Preparation and Functional Analysis of Highly Reproducible, Large, Uniform Spheroids and Organoids.

Growing cells as 3D structures need not be difficult. Often, it is not necessary to change cell type, additives or growth media used. All that needs to be changed is the geometry: cells (whether primary, induced pluripotent, transformed or immortal) simply have to be grown in conditions that promote cell-cell adhesion while allowing gas, nutrient, signal, and metabolite exchange. Downstream analysis can become more complicated because many assays (like phase contrast microscopy) cannot be used, but their replacements have been in use for many years. Most importantly, there is a huge gain in value in obtaining data that is more representative of the organism in vivo. It is the goal of the protocols presented here to make the transition to a new dimension as painless as possible. Grown optimally, most biopsy derived organoids will retain patient phenotypes, while cell (both stem cell, induced or otherwise or immortalized) derived organoids or spheroids will recover tissue functionality.

Elucidating the cellular response of silver nanoparticles as a potential combinatorial agent for cisplatin chemotherapy.

BACKGROUND: Combination chemotherapy uses drugs that target different cancer hallmarks, resulting in synergistic or additive toxicity. This strategy enhances therapeutic efficacy as well as minimizes drug resistance and side effects. In this study, we investigated whether silver nanoparticles act as a combinatorial partner to cisplatin. In so doing, we compared post-exposure biological endpoints, intracellular drug accumulation, and changes in the proteome profile of tumoral and normal cell lines. RESULTS: Combinatorial exposure corresponded to cytotoxicity and oxidative stress in both cell lines, yet was substantially more effective against tumoral cells. Proteome analysis revealed that proteins related to energy metabolism pathways were upregulated in both cell lines, suggesting that combinatorial exposure corresponded to energetic modulation. However, proteins and upstream regulators involved in the cell cycle were downregulated, indicating reduced cell proliferation. The response to oxidative stress was markedly different in both cell lines; downregulation of antioxidant proteins in tumoral cells, yet upregulation of the antioxidant defense system in normal cells. These outcomes may have avoided higher cell death rates in normal cells. CONCLUSIONS: Taken together, our results indicate that combining silver nanoparticles with cisplatin increases the biological activity of the latter, and the combination warrants further exploration for future therapies.

Response to and recovery from treatment in human liver-mimetic clinostat spheroids: a model for assessing repeated-dose drug toxicity.

Medicines are usually prescribed for repeated use over shorter or longer times. Unfortunately, repeated-dose animal toxicity studies do not correlate well with observations in man. As emphasized by the '3Rs' and the desire to phase-out animal research, in vitro models are needed. One potential approach uses clinostat-cultured 3D HepG2-C3A liver-mimetic spheroids. They take 18 days to recover in vivo physiological functionality and reach a metabolic equilibrium, which is thereafter stable for a year. Acute and chronic repeated-dose studies of six drugs (amiodarone, diclofenac, metformin, phenformin, paracetamol and valproic acid) suggest that spheroids are more predictive of human in vivo toxicity than either 2D-cultured HepG2 cells or primary human hepatocytes. Repeated non-lethal treatment results in a clear response and return to equilibrium. Mitochondrial toxic compounds can be identified using a galactose-based medium. Some drugs induced a protective (or stress) response that intensifies after the second treatment. This 3D spheroid model is inexpensive, highly reproducible and well-suited for the determination of repeated-dose toxicity of compounds (naturally or chemically synthesized).

Silver nanoparticle-induced expression of proteins related to oxidative stress and neurodegeneration in an in vitro human blood-brain barrier model.

Silver nanoparticles (AgNPs) have been reported to penetrate the central nervous system (CNS) and induce neurotoxicity. However, there is a paucity of understanding of the toxicity of AgNPs and their effect on the blood-brain barrier (BBB) including the underlying molecular mechanism(s) of action. Such information is important for the formulation of new strategies for delivery of biological therapeutics to central nervous system (CNS) targets. Using an in vitro BBB model and mass spectrometry-based proteomics, we investigated alterations in the proteomes of brain endothelial cells and astrocytes at different time points after AgNPs exposure (24 and 48 h). Our data showed that several proteins involved in neurodisorders and neurodegeneration were significantly upregulated in endothelial cells (e.g. 7-dehydrocholesterol reductase, zinc transporters 1 and 6), while proteins responsible for maintaining brain homeostasis were significantly downregulated (e.g anti-oxidative proteins glutathione peroxidase 1 and glutathione peroxidase 4). Many inflammatory pathways were significantly upregulated at 24 h post-AgNPs exposure (C9 pathway), while at 48 h proteins involved in BBB damage and anti-inflammatory responses were upregulated (quinoneoxidoreductase1 and glutamate cysteine ligase catalytic subunit) suggesting that by the later time point, cellular protection pathways had been activated to rescue the cells from AgNPs-induced toxicity. Our study suggests that in the initial stage of exposure, AgNPs exerted direct cellular stress on the endothelial cells by triggering a pro-inflammatory cascade. This study provides detailed insight into the toxic potency of AgNPs on in vitro BBB model and adds to the understanding of the adaptive role of BBB with regards to AgNPs-mediated toxicity.

Co-exposure to silver nanoparticles and cadmium induce metabolic adaptation in HepG2 cells.

Although multiple studies have reported the toxicological effects and underlying mechanisms of toxicity of silver nanoparticles (AgNP) in a variety of organisms, the interactions of AgNP with environmental contaminants such as cadmium are poorly understood. We used biochemical assays and mass spectrometry-based proteomics to assess the cellular and molecular effects induced by a co-exposure of HepG2 cells to AgNP and cadmium. Cell viability and energy homeostasis were slightly affected after a 4-h exposure to AgNP, cadmium, or a combination of the two; these endpoints were substantially altered after a 24-h co-exposure to AgNP and cadmium, while exposure to one of the two contaminants led only to minor changes. Proteomics analysis followed the same trend: while a 4-h exposure induced minor protein deregulation, a 24-h exposure to a combination of AgNP and cadmium deregulated 43% of the proteome. The toxicity induced by a combined exposure to AgNP and cadmium involved (1) inactivation of Nrf2, resulting in downregulation of antioxidant defense and proteasome-related proteins, (2) metabolic adaptation and ADP/ATP imbalance, and (3) increased protein synthesis possibly to reestablish homeostasis. The adaptation strategy was not sufficient to restore ADP/ATP homeostasis and to avoid cell death.

Mobilization of CD34+CXCR4+ stem/progenitor cells and the parameters of left ventricular function and remodeling in 1-year follow-up of patients with acute myocardial infarction.

Mobilization of stem cells in acute MI might signify the reparatory response. Aim of the Study. Prospective evaluation of correlation between CD34+CXCR4+ cell mobilization and improvement of LVEF and remodeling in patients with acute MI in 1-year followup. Methods. 50 patients with MI, 28 with stable angina (SAP), and 20 individuals with no CAD (CTRL). CD34+CXCR4+ cells, SDF-1, G-CSF, troponin I (TnI) and NT-proBNP were measured on admission and 1 year after MI. Echocardiography and ergospirometry were carried out after 1 year. Results. Number of CD34+CXCR4+ cells in acute MI was significantly higher in comparison with SAP and CTRL, but lower in patients with decreased LVEF ≤40%. In patients who had significant LVEF increase ≥5% in 1 year FU the number of cells in acute MI was significantly higher versus patients with no LVEF improvement. Number of cells was positively correlated (r = 0,41, P = 0,031) with absolute LVEF change and inversely with absolute change of ESD and EDD in 1-year FU. Mobilization of CD34+CXCR4+ cells in acute MI was negatively correlated with maximum TnI and NT-proBNP levels. Conclusion. Mobilization of CD34+CXCR4+ cells in acute MI shows significant positive correlation with improvement of LVEF after 1 year.

Relationship between R-R interval variation and left ventricular function in sinus rhythm and atrial fibrillation as estimated by means of heart rate variability fraction.

BACKGROUND: Reduced heart rate variability (HRV) is associated with a poor outcome in patients with sinus rhythm (SR) or atrial fibrillation (AF). However, cut-off points for HRV measures differ between SR and AF. We hypothesized that a global index of 24-hour HRV based on evaluation of scatterplot would describe HRV irrespective of cardiac rhythm. METHODS: 407 patients with ischemic heart disease (317 male, 90 female, mean age 57 ± 9 years) were studied. 331 patients had SR and 76 patients had AF. 24-hour ECGs were recorded, and standard HRV indices were calculated. Scatterplots was used to determine the HRV fraction (HRVF, %). HRV measures were compared in respect to left ventricular ejection fraction (LVEF £ 35% or 〉 35%). RESULTS: Standard HRV measures were higher in AF-patients despite the mean RR interval was lower. In patients with LVEF £ 35%, standard HRV indices were lower in SR group, in AF group only SDNN and RMSSD were reduced. The HRVF was comparably reduced (SR 39.3 ± 15.3%, AF 37.3 ± 17.9%). In patients with LVEF 〉 35%, HRVF did not differ between SR (47.2 ± ± 10.5%) and AF (46.1 ± 12.1%). The HRVF correlated with SDNN and SDANN (~0.85) in SR. Correlations were weaker in AF (~0.6). Standard HRV indices and HRVF showed similar relations with LVEF, but only in AF at the same range. CONCLUSIONS: The HRV fraction allows for HRV evaluation irrespective of cardiac rhythm. The index elicited a similar dependence of HRV on left ventricular function in SR and AF.

[Massive pulmonary embolism without echocardiographic features of right ventricular overload]. / Masywny zator tetnicy plucnej bez cech przeciazenia prawej komory w badaniu echokardiograficznym.

A case of 70-year-old patient with massive pulmonary embolism confirmed in CT, but without changes in right ventricle size and function in echocardiography is presented. This case is consistent with literature data that echocardiography has relatively low sensitivity in the diagnosis of acute pulmonary embolism.

The prognostic role of electrocardiographic left ventricular mass assessment for identifying PCI-treated patients with acute ST-elevation myocardial infarction at high risk of unfavourable outcome.

BACKGROUND: In prognostic terms, evaluation of an ECG recording in acute myocardial infarction (AMI) appears to be inferior to echocardiographic (ECHO) assessment of left ventricular remodelling and the activities of cardiac enzymes and certain hormones. It was our hypothesis that, in the era of interventional treatment of AMI, some ECG parameters are still valid for the purpose of risk stratification. METHODS: A total of 66 consecutive patients with AMI (43 male and 23 female, with a mean age of 61 +/- 11 years) were treated with primary percutaneous coronary intervention (PCI). In each patient ECG and ECHO examinations were performed within 5-7 days of admission for the detection of left ventricular hypertrophy (LVH). In further analysis the following ECG- based LVH parameters were taken into consideration: Sokolov-Lyon voltage duration (SLVd), Cornell voltage duration CVd), 12-lead QRS voltage duration (12QRSVd), their product with QRS duration and an ECG index of left ventricular mass (LVMI(ECG)). Patients were followed for 6 months. The combined end-point included death, infarction, a need for prompt coronary intervention and hospitalization for heart failure. RESULTS: The combined end-point was observed in 16 patients (24.2%). Survival analysis revealed that the most important prognostic factors were associated with a prolongation of the QRS duration. Increased SLVd was found in 43% of the patients with events compared to 14% in those without them (p < 0.01), CVd in 43% vs. 12% (p < 0.05), 12QRSVd in 81% vs. 44% (p < 0.05) and LVMI(ECG) in 75% vs. 26%, p < 0.001). There was no evidence for a difference in Cornell voltage. Univariate logistic regression indicated a 4-fold to 8-fold increase in the risk of events associated with abnormal SLV, SLVd or LVMI(ECG). Multivariate Cox analysis showed that the LVH presence in the ECG, defined as an increased SLVd product or increased LVMI(ECG), was an independent predictor of cardiovascular events after AMI. CONCLUSIONS: In the era of interventional treatment of AMI, the ECG features of left ventricular hypertrophy carry independent significant prognostic information. (Cardiol J 2007; 14: 347-354).

Left ventricular mass and hypertrophy assessment by means of the QRS complex voltage-independent measurements.

UNLABELLED: ECG QRS-complex voltage-based criteria are relatively insensitive for detection of increased left ventricular mass (LVM). We developed and evaluate a new ECG index for LV hypertrophy (LVH) detection regardless of the QRS voltage. METHODS: Study population consisted of 106 patients (73 m, 33 f, aged 60 +/- 10 years) with established coronary artery disease (CAD). All patients had LVM assessed echocardiographically and indexed to BSA (LVMI(ECHO)). LVH was diagnosed if LVMI(ECHO) >117 g/m2 in men and >104 g/m2 in women. LV geometry was also determined. Analysed ECG variables, obtained from 12 leads recorded simultaneously, were: the QRS complex duration (QRSd, ms), the average 12-lead time to maximal deflection (TMD, ms), the average 12-lead QRS complex voltage (12QRSV, mV), the average product of 12 lead QRS voltage and duration (12QRSVd, mV ms), Sokolow-Lyon voltage and V-d product (SLV, SLVd), Cornell voltage and V-d product (CV, CVd). A newly developed index, LVM(ECG), was calculated, as LVM(ECG) = [(2 x TMD+QRSd/pi)3-(QRSd/pi)3]*0.0001 (ms3), and indexed to BSA (LVMI(ECG), ms3/m2). RESULTS: Means of the QRS voltage-related parameters were similar in patients with LVH and normal LVM. Greater differences existed between both groups when the QRS voltage-duration products were compared. LVMI(ECG) was most powerful in distinguishing between groups (130 +/- 33 LVH vs 91 +/- 21 normal LVM, p < 0.001). LVMI(ECG) correlated with LVMI(ECHO) better (r = 0.77, p < 0.001) than other indices (r coefficients between 0.24 for SLV and 0.49 for CVd). None of the examined indices allowed for distinction between eccentric and concentric LVH. The new index showed better statistical performance (area under ROC = 0.861) compared to the other indices (AUC range 0.545-0.697, p<0.001 vs LVMI(ECG)). At the specificity level of 92%, the value of LVMI(ECG) > 120 ms3/m2 had the sensitivity of 64% for detection of increased LVM. The sensitivities of the other parameters were significantly lower (sensitivity range 18-42%). Relative intra- and interobserver errors and correlation coefficients for LVMI(ECG) calculation were 0.4% and 1.6% and r = 0.94 and 0.98, respectively. CONCLUSIONS: In patients with CAD an assessment of LV mass and detection of hypertrophy using the QRS complex time-dependent index is feasible. The new index correlated well with echocardiographically-determined LVM and showed better statistical performance than indices which include QRS-voltage measurements. The results are promising and warrant further studies to evaluate the utility of the new index as a risk predictor.

Increased variability of the coupling interval of premature ventricular beats may help to identify high-risk patients with coronary artery disease.

OBJECTIVE: The purpose of this study was to determine the characteristics and predictive value of the variability of coupling interval of ventricular premature beats (VPBs) for cardiac mortality in patients with coronary artery disease (CAD). BACKGROUND: Frequent VPBs have been linked to an increased risk for cardiac death in patients with coronary artery disease. It is unknown whether analysis of coupling interval of VPBs from ambulatory ECG recordings can be used for risk statification in these patients. METHODS: In 78 consecutive symptomatic patients with documented CAD who presented with frequent VPBs (>720/24 h), the analysis of VPBs' coupling interval (SDNV) was performed. Left ventricular function, ventricular arrhythmias and simple measures of heart rate variability were assessed. Mean follow-up was 702+/-329 days. Cardiac mortality was the primary end-point of the study. RESULTS: During follow-up, 14 patients died-11 deaths were cardiac. Left ventricular ejection fraction (LVEF)<40%; no beta-blocker treatment and digoxin use were clinical variables showing a significant association with cardiac mortality. The presence of non-sustained ventricular tachycardia (nsVT), especially if more than five episodes were present; short mean sinus cycle (<750 ms) and SDNV were associated with cardiac deaths. Mean SDNV was 79+/-29 in victims and 63+/-29 in survivors (p<0.05). Univariate Cox regression analysis revealed that the presence of SDNV>80 ms carried a relative risk of 6.7 for cardiac mortality. The adjusted relative risk was 13.3 for nsVT and 4.4 for SDNV>80 ms. Among patients with nsVT, mortality rate was significantly higher with SDNV>80 ms (58%), compared to lower SDNV (14%, p<0.01). Sixty-four percent mortality rate was observed in patients with LVEF<40%, presence of nsVT and SDNV>80 ms, compared to 17% in similar patients with lower SDNV (p<0.05). CONCLUSION: The analysis of coupling interval of ventricular premature beats form the same 24-h ECG recordings may complement the standard Holter analysis for risk stratification. This seems especially promising in the subgroups of patients at highest risk-those with LV systolic dysfunction, non-sustained VT or both.

[Heart rate variability in patients with hypertrophic cardiomyopathy and in their close relatives]. / Zmiennosc rytmu serca u chorych z kardiomiopatia przerostowa i ich bliskich krewnych.

The results of HRV analysis in patients with hypertrophic cardiomyopathy (HCM) are conflicting. We evaluated short-term HRV in patients with familiar HRV and in their close relatives. 31 families of patients with HCM were examined. There were 43 patients with HCM confirmed by 2D-echo (HCM-group, 23 f, 20 m, aged 46 +/- 14 ys), and 157 family members (REL-group, 75 f, 82 m, aged 29 +/- 17 ys). The control group consisted of 180 healthy subjects (80 f, 100 m, aged 33 +/- 12 ys). In each subject 512 consecutive sinus beats were recorded in supine position during spontaneous breathing using computer-assisted amplifier (A/D 12 bit, 1 kHz). Mean RR interval (RRI, ms), its standard deviation (SDRR, ms) and spectral measures (FFT, Blackman-Harris window): PSD of high frequency (HF) and low frequency (LF) [ms2/Hz], as well as respiratory rate (BPM) were measured. Patients with HCM had significantly shorter RRI (867 +/- 121) as compared to the controls (919 +/- 138, p < 0.05). The RRI was also shorter in the relatives (851 +/- 150, p < 0.01). In HCM and REL groups the respiratory rate was faster (16.7 +/- 3.0 and 17.1 +/- 3.4, respectively), as compared to the controls (14.5 +/- 2.9, both p < 0.01). The HRV measures were reduced in the HCM-group (SDRR 31.4 +/- 10.6, lnHF 7.71 +/- 0.65, lnLF 8.22 +/- 0.65 and LF/HF 1.07 +/- 0.10), as compared to the controls (SDRR 64.8 +/- 23.9, lnHF 8.79 +/- 0.61, lnLF 8.87 +/- 0.65, all p < 0.001 and LF/HF 1.01 +/- 0.07, p < 0.01). In the REL-group SDRR and lnHF were significantly reduced (SDRR 52.4 +/- 24.1, lnHF 8.48 +/- 0.78, p < 0.001), while the remaining parameters were comparable. The HRV reduction was more expressed in HCM-patients and family-members < 30 years of age. A significantly reduced age-, sex- and RRI-adjusted SDNN was observed in 54% pts in HCM-group and in 42% subjects in members-group. Reduced heart rate variability is frequently seen not only in patients with diagnosed HCM, but also in a substantial number of their kindred.