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Cell and Tissue Culture

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Cell and tissue culturing

The Cell and Tissue Culture Core Facility provides the opportunity to design, prepare (thaw, passage and handle specific cell lines), perform experiments and evaluate results based on personalized cell and tissue cultures using its comprehensive instruments and equipments. In addition to the experimental set-up based on two-dimensional cell and tissue cultures, users have the option to set up an experimental model based on complex cell and tissue cultures. Users are also provided with the ability to dissociate primary cells from animal or human tissues or to separate a particular cell population from body fluids.

The available cell cultures are cultured in all cases at 37 °C incubators with 5% CO2 and high humidity. The experiments are performed in a laminar flow cabinet under sterile conditions. In addition to the instruments highlighted below, we have the essential instruments and tools needed for cell and tissue culture techniques, such as:

  • Microscope for monitoring cell cultures (EVOS XL Core Imaging System)
  • Incubators
  • Cell culture hoods (even for cytostatic treatment)
  • Nitrogen tank suitable for long-term storage of our cell lines
  • Different types of cell culture media and supplements


Isolation of extracellular vesicles

Extracellular vesicles (EVs) are membrane-delimited particles released from all type of cells into the extracellular space. According to their size and biogenesis EVs can be differentiated into three subgroups such as exosomes, microvesicles and apoptotic bodies. These vesicles can be isolated from many body fluids (blood, urine, breast milk etc.) or from cell culture supernatant. Apoptotic bodies and microvesicles can be elutriated using high-speed centrifugation, but exosomes require PEG-based isolation method.

We are able to isolate exosomes from the following fluids:

  • blood serum
  • blood plasma
  • urine
  • cell culture supernatant
  • other body fluid (e.g. milk)


Equipments

NanoSight NS300

The Malvern Panalytical NanoSight NS300 Instrument provides an easy-to-use, reproducible platform for nanoparticle characterization, especially size characterization of extracellular vesicles. Particles in liquid suspension are loaded into a sample chamber. In the path of the beam the particles scatter the laser light which is easily collected by the microscope objective and is viewed with a digital camera. The camera captures a video of the particles moving under Brownian motion. The NS300 allows rapid analysis of the size distribution and concentration of all types of nanoparticles from 10 nm - 2 µm in diameter, depending on the instrument configuration and sample type. With the ability to be supplied with interchangeable laser modules and the introduction of a motorized filter wheel means different fluorescent labels can be analyzed.

Detectable particle size: 10nm - 2000nm

Lasers: 488nm (Blue), 532nm (Green)

Concentration: 109 particle / ml

NS300 can be applied in the development of different delivery system studies:

  • Viral vaccine research
  • Nano-toxicology and viral vaccine detection
  • Protein aggregation studies
  • Extracellular vesicle characterization in different stages of diseases
  • Exosome and microvesicle characterization


QuantStudio™ 12K Flex Real-Time PCR System

The QuantStudio™ 12K Flex Real-Time PCR system takes qPCR technology to a next level with maximum throughput and minimal resources. Using a simple workflow gene expression analysis or miRNA profiling can be conducted with reduced start-up and hands-on time. The results can be easily analyzed using ExpressionSuite Software, an easy-to-use data-analysis tool that utilizes the comparative Cτ (ΔΔCτ) method to rapidly and accurately quantify relative gene expression across a large number of genes and samples.

Fluorophore compatibility: Taqman and SYBR Green reagents

Interchangeable blocks and plate compatibility: TaqMan Array Card (384-well)

                                                                            Fast 96-well plate



GentleMACS Octo Dissociator

The gentleMACS™ Octo Dissociator is a benchtop instrument for semi-automated and standardized tissue dissociation or homogenization of up to eight samples. Single-cell suspensions or thorough homogenates from virtually any tissues are easily and reproducibly obtained using the unique C Tubes or M Tubes. These tubes allow sample preparation in a closed and sterile environment with minimized cross-contamination.

Optimized, pre-set gentleMACS Octo Dissociator Programs for easy dissociation of tissues into single-cell suspensions:

  • mouse or human tumor
  • mouse or rat neonatal heart
  • neural tissue
  • mouse spleen, lung, lamina propria, muscle, epidermis, or liver
  • mouse or human skin


NanoShuttle™ technology

Greiner Bio-One's magnetic cell culturing technology uses biocompatible nanoparticles (NanoShuttle™-PL) in order to magnetize cells. After magnetization cells can form three-dimensional spheroid models in a relatively short period of time (24 hours). This high throughput technique is compatible with fluorescent microscopy, Western blot, qRT-PCR, flow cytometry, viability assays and chemiluminescence methods.

The advantages of magnetic cell culture include:

  •         Mimicking native tissue environment
  •         Rapid 3D model formation within hours
  •         No specialized equipment, media, or artificial substrate
  •         Easy to handle / no sample loss
  •         Allows co-culture

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OctoMACS Separator

The OctoMACS™ Separator contains a powerful permanent magnet that induces a high-gradient magnetic field to retain cells labeled with even small amounts of MACS MicroBeads. It allows the performance of up to eight simultaneous separations in combination with different MACS Columns:

  • MS Columns for positive selection or depletion of cells
  • Large Cell Columns for positive selection of large cells, e.g., megakaryocytes
  • M Columns for isolation of molecules, e.g., RNA

 



MPW-223c laboratory centrifuge

Based on a cytospin method cells in cell suspension can be easily concentrated onto glass slides in order to perform different  staining methods. The MPW-223c laboratory centrifuge is perfectly suitable to carry out such techniques.



LAS-4000 image analyzer

The LAS 4000 is a camera system for producing digital images of chemiluminescent or fluorescent gel and membrane samples. The digital image allows the subsequent molar mass or quantitative analysis for e. g. Western blot.

Detection methods:

  • luminescence
  • fluorescence

Exchangeable light sources and filters:

  • infrared
  • red
  • green
  • blue
  • UV


Quantstudio 3D Digital PCR

The QuantStudio 3D Digital PCR system is a chip-based technology which allows a simple and streamlined workflow with the ability to thermal cycle up to 24 chips at one time in less than 30 seconds to read each chip. The QuantStudio 3D Digital PCR System produces absolute quantification data, concentration measurements in copies/μL without a standard curve.

Applications:

  • Rare allele detection
  • Precise copy number variation
  • Absolute quantification of viral load
  • Absolute quantification of nucleic acid standards
  • Absolute quantification of next‐generation sequencing libraries
  • Low‐level pathogen detection
  • Low‐level fold change of gene expression

Applied Biosystems™ QuantStudio™ 3D AnalysisSuite™ Cloud Software is used for the analysis of data derived from the QuantStudio 3D Digital PCR instrument. The software also enables analysis of QuantStudio 3D chips and accurate, highly precise measurements of genomic targets in copies per microliter. Moreover it allows low-fold detection comparison and ratio analysis of genomic targets, as well as copy number variation. Multiple chips can be combined and analyzed in a single project.



Dr. Krisztián Kvell
associate professor
krisztian.kvell@aok.pte.hu
29250
Dr. Krisztina Bánfai
assistant lecturer
banfai.krisztina@pte.hu
29250
Dr. Mohamed Mahmoud Abdelwahab ElHusseiny
assistant lecturer
elhusseiny.mohamed@pte.hu
29250
Kitti Garai
assistant lecturer
garai.kitti@pte.hu
29250
Judit Bóvári-Biri
research fellow
bovari.judit@pte.hu
29250

2021

Publications

Soltani A, Kajtar B, Abdelwahab EHMM, Steib A, Horvath Z, Mangel L, Jaromi L, Pongracz JE. Is an Immunosuppressive Microenvironment a Characteristic of Both Intra- and Extraparenchymal Central Nervous Tumors? Pathophysiology (2021) 28:34–49. doi:10.3390/pathophysiology28010004.

 

2020

Publications

Boda F, Banfai K, Garai K, Kovacs B, Almasi A, Scheffer D, Sinkler RL, Csonka R, Czompoly T, Kvell K. Effect of Bitis gabonica and Dendroaspis angusticeps snake venoms on apoptosis-related genes in human thymic epithelial cells. J Venom Anim Toxins Incl Trop Dis. 2020 Dec 14;26:e20200057. doi: 10.1590/1678-9199-JVATITD-2020-0057. PMID: 33402885; PMCID: PMC7745260.

Papp H, Zeghbib S, Földes F, Banfai K, Madai M, Kemenesi G, Urbán P, Kvell K, Jakab F. Crimean-Congo hemorrhagic fever virus infection triggers the upregulation of the Wnt signaling pathway inhibitor genes. Virus Genes. 2020 Aug;56(4):508-514. doi: 10.1007/s11262-020-01759-z. Epub 2020 Apr 25. PMID: 32335793.

Papp E, Steib A, Abdelwahab EM, Meggyes-Rapp J, Jakab L, Smuk G, Schlegl E, Moldvay J, Sárosi V, Pongracz JE. Feasibility study of in vitro drug sensitivity assay of advanced non-small cell lung adenocarcinomas. BMJ Open Respir Res. 2020 Jun;7(1):e000505. doi: 10.1136/bmjresp-2019-000505. PMID: 32527872; PMCID: PMC7292226.

Kiss E, Abdelwahab EHMM, Steib A, Papp E, Torok Z, Jakab L, Smuk G, Sarosi V, Pongracz JE. Cisplatin treatment induced interleukin 6 and 8 production alters lung adenocarcinoma cell migration in an oncogenic mutation dependent manner. Respir Res (2020) 21:120. doi:10.1186/s12931-020-01389-x

2019

Publications

Abdelwahab, EMM; Rapp, J; Feller, D; Csongei, V; Pal, S; Bartis, D; Thickett, DR; Pongracz JE.: Wnt signaling regulates trans-differentiation of stem cell like type 2 alveolar epithelial cells to type 1 epithelial cells. Respiratory Research 20:1, 9 p. (2019)

Garai, K; Adam, Z; Herczeg, R; Katai, E; Nagy, T; Pal, S; Gyenesei, A; Pongracz, JE; Wilhelm, M; Kvell, K.: Artificial Neural Network Correlation and Biostatistics Evaluation of Physiological and Molecular Parameters in Healthy Young Individuals Performing Regular Exercise. Frontiers in Physiology 10 Paper: 1242, 11 p (2019)

Vas V, Háhner T, Kudlik G, Ernszt D, Kvell K, Kuti D, Kovács KJ, Tóvári J, Trexler M, Merő BL, Szeder B, Koprivanacz K, Buday L. Analysis of Tks4 Knockout Mice Suggests a Role for Tks4 in Adipose Tissue Homeostasis in the Context of Beigeing. Cells. 2019 Aug 5;8(8). pii: E831. doi: 10.3390/cells8080831.

Banfai K, Ernszt D, Pap A, Bai P, Garai K, Belharazem D, Pongracz JE, Kvell K. ‚Beige‘ Cross Talk between the Immune System and Metabolism. Front. Endocrinol.2019 May 24.  doi: 10.3389/fendo.2019.00369.7       

Banfai K, Garai K, Ernszt D, Pongracz JE, Kvell K. Transgenic Exosomes for Thymus Regeneration. Front Immunol. 2019 Apr 24;10:862. doi: 10.3389/fimmu.2019.00862.

Abdelwahab EMM, Pal S, Kvell K, Sarosi V, Bai P, Rue R, Krymskaya V, McPhail D, Porter A, Pongracz JE. Mitochondrial dysfunction is a key determinant of the rare disease lymphangioleiomyomatosis and provides a novel therapeutic target. Oncogene. 2019 Apr;38(16):3093-3101. doi: 10.1038/s41388-018-0625-1. Epub 2018 Dec 20.

2018

Publications

Feller D, Kun J, Ruzsics I, Rapp J, Sarosi V, Kvell K, Helyes Z, Pongracz JE. Cigarette Smoke-Induced Pulmonary Inflammation Becomes Systemic by Circulating Extracellular Vesicles Containing Wnt5a and Inflammatory Cytokines. Front Immunol. 2018 Jul 25;9:1724. doi: 10.3389/fimmu.2018.01724. eCollection 2018.

Boda F, Banfai K, Garai K, Curticapean A, Berta L, Sipos E, Kvell K. Effect of Vipera ammodytes ammodytes Snake Venom on the Human Cytokine Network. Toxins (Basel). 2018 Jun 25;10(7). pii: E259. doi: 10.3390/toxins10070259.

2017

Publications

Ernszt D, Banfai K, Kellermayer Z, Pap A4 Lord JM, Pongracz JE, Kvell K.PPARgamma Deficiency Counteracts Thymic Senescence. Front Immunol. 2017 Nov 6;8:1515. doi: 10.3389/fimmu.2017.01515. eCollection 2017.

Pénzes Á, Mahmud Abdelwahab EM, Rapp J, Péteri ZA, Bovári-Biri J, Fekete C, Miskei G, Kvell K, Pongrácz JE. Toxicology studies of primycin-sulphate using a three-dimensional (3D) in vitro human liver aggregate model. Toxicol Lett. 2017 Nov 5;281:44-52. doi: 10.1016/j.toxlet.2017.09.005.

Rapp J, Jaromi L, Kvell K, Miskei G, Pongracz JE. WNT signaling - lung cancer is no exception. Respir Res. 2017 Sep 5;18(1):167. doi: 10.1186/s12931-017-0650-6.

Vesel M., Rapp J., Feller, D., Kiss, E., Jaromi, L., Meggyes, M, Miskei, G., Duga, B., Smuk, G., Laszlo, T., Karner, I., Pongracz, J.E.: ABCB1 and ABCG2 drug transporters are differentially expressed in non-small cell lung cancers (NSCLC) and expression is modified by cisplatin treatment via altered Wnt signaling. 2017. Respiratory Research 18:52 DOI 10.1186/s12931-017-0537-6

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