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LABORATORIES AND RESEARCH SERVICES

What are our distinctive features?

LABORATORIES AND RESEARCH SERVICES

With its experienced specialist team and unique research infrastructure, the Jagiellonian Center of Innovation performs a broad range of research in the field of medicine, biotechnology, biology, chemistry, pharmacy, cosmetology and environmental protection.

The Jagiellonian Center of Innovation offers services in the field of:• Biological analysis• Chemical analysis• Structural analysis and Medicine Formulation analysis• Surface analysis • Medical diagnostics• Tissue engineering

Unique research infrastructure

Full range offering:we perform services ranging

from the simplest studies to complex research projects

Comprehensive implementation of research

advice upon study development – budget – performance – result interpretation

BIOLOGICAL ANALYSIS

Biochemical analysis of proteins – The Jagiellonian Center of Innovation offers a wide range of analytical services in proteomics, based on electrophoretic, chromatographic and mass spectroscopy methods.

Electrophoretic analysesBiochemical analysis of proteins using electrophoretic methods, including SDS-PAGE 2D analysis, native electrophoresis and Western Blott.

Chromatographic analyses (WYATT)In the proteomic laboratory of the Jagiellonian Center of Innovation, we can optimize and perform protein separations using SEC-MALS and SEC-MALS-QELS methods, with Heleos II and DynaProNanoStar detectors. The detectors allow for determining mass and concentration without the necessity to use a reference. Moreover, with a DLS detector we can work in a cuvette mode and hence determine (at volumes of the order of several µl) various factors quickly, including: • relative and absolute molecular weight• determination of protein crystallization parameters• determination of protein melting point• measurement of conformation changes in the native environment (via change in hydration radius Rh parameter)

Mass spectrometry (QTOF)The Jagiellonian Center of Innovation performs spectroscopic analyses using Impact II QTOF (Bruker) device, with optional connection to HPLC chromatograph. This allows for the generation of high-resolution mass spectra (HRMS) and low fragmentation of the studied molecules. The use of ProteinScape base and BioTools software (Bruker) included in the set enables:• automation of the spectra base search process• determination of protein structures • de novo protein sequencing along with optimization of results from very big matrices

Analysis of intermolecular interactions Based on MST and SPR methods using Monolith NT.115, Biacore X-100 or ELISA test, the Jagiellonian Center of Innovation offers analyses of intermolecular interactions, such as:• protein-protein interactions• protein-nucleic acid interactions• protein-lipid, protein-sugar interactions• protein-low molecular weight compounds interactions

MST (Monolith NT.115)A method based on a microscale thermophoresis (MST) phenomenon that describes the mobility of molecules in a solution with a temperature gradient. This unique technique is based on the use of the phenomenon of molecule movement change in temperature gradient, which allows for:• analyzing protein interactions in solution • making the analysis independent of the chemical composition of

the solution • the option to perform experiments in conditions similar to

native ones

SPR (Biacore X-100) The Surface Plasmon Resonance (SPR) phenomenon makes it possible to determine the strength of intermolecular interactions in real time. The SPR phenomenon is based on oscillation of conducting electrons present on the biosensor due to the incident light, which results in absorption of a strictly defined amount of the incident light wave, leading to a decrease in the energy of the reflected wave. The method also allows for:• determination of the studied analyte concentration• studying the affinity of the studied intermolecular interactions • analysis of kinetics and thermodynamics of the studied system• determination of association and dissociation constants of the complex formed upon the interaction

ELISA testsThis method is used for the detection of proteins in the studied material, using antibodies. Antibodies are conjugated with an enzyme that (upon the addition of the appropriate substrate) catalyzes the reaction of formation of a spectrophotometrically assayed product. The method is commonly used for scientific and diagnostic processes, such as:• determination of concentration and type of proteins in the studied sample• diagnostics of viral and bacterial presence• diagnostics of autoimmunological and neoplastic diseases

PCR, qPCR, RT-PCRThe Polymerase Chain Reaction (PCR) is a method that allows for the multiplication of specific DNA fragments in vitro. Real Time-PCR is a quantitative PCR reaction and it enables simultaneous multiplication of chosen DNA fragments and monitoring the amount of products formed in each cycle. The Jagiellonian Center of Innovation offers services within the scope of optimization of PCR and Real Time PCR reactions using a non-specific SYBR Green method or a specific method with complementary oligonucleotide probes, based on a fluorescence resonance energy transfer (FRET) between the molecules of a donor and a quenching agent. Additionally, we also offer RT-PCR (reverse transcription PCR), where RNA is used as a matrix.

The PCR technique has many applications mainly in genome studies, characterization of gene expression, gene cloning and clinical diagnostics.

Contact: dr Łukasz Kutrzeba T: +48 12 297 46 27 K: +48 515 075 500 E: [email protected] WWW.JCI.PL You are also invited to cooperate with us in the scope of funds acquisition and research grant projects.

CHEMICAL ANALYSIS

Chromatographic analysis – the technique of substance separation where the studied mixture flows through a column filled with an appropriate bed and is separated by obtaining equilibrium of each analyte between the stationary and the mobile phase.

Liquid chromatographyThe Quality Control Laboratory of the Jagiellonian Center of Innovation is equipped with analytical uHPLC systems (Dionex) and sets for preparative purification of chemical substances, combined with the following detectors:• UV – VIS „DAD” (190-900nm), analytical and semi-micro cell• ELSD• CoronaVeo RS (working temperature range +5°C to +100°C)• MALS-QELS ((mass can be determined in a range from 100Da to 1250kDa)• Q-ToF – (MS/MS), (ESI, APCI) spectral range from 20 to 15000m/z)• Fluorescence FLD (spectral range: Ex from 200 to 630; Em 220 to 650)• Refractometric RI• LC PureFlash(Interchim) with UV-VIS „DAD” and ELSD detectors, with an automated fraction collector• Preparative set LC-MS (Shimadzu) with UV (from 190 to 370nm), ELSD and MS („single quadrupole”) detectors

The instruments allow for:• broad analytical possibilities• significantly shorter analysis time thanks to the use of UHPLC systems• possibility to purify 10 g of substance per analysis• detection of substances invisible in UV-VIS range, owing to Corona and ELSD detectors• cheap and repeatable substance purification via separation

Gas chromatography• chromatographic sets (Shimadzu) with autosampler for injection of liquid samples and HeadSpace attachment • detectors: flame-ion detector FID for assaying volatile compounds, MS detector

The instruments enable:• analysis of volatile substances collected from headspace using the Head Space adapter • easier qualitative and quantitative analysis of unknown substances (built-in mass library)

Supercritical fluid chromatography (CO2) (SFC):• chromatographic sets (Waters) with an analytical system• PDA detectors for the optimization of separation methods• fully automated preparative set combined with PDA (190 to

900 nm) and MS („single quadrupole”)

The instruments allow for:• significantly shorter analysis time for chiral compounds• possibility to separate achiral compounds that cannot be

separated using the conventional chromatographic methods due to their properties (high polarity, lability)

• separation of optically-active compounds owing to a set of chromatographic columns

Assaying Total Organic Carbon content, TOC: Device (GE Sievers 9M) with a measurement range of 0.03 ppb-50 ppm.

The measurement is performed via chemical oxidation of organic substances and then measurement of conductance in the obtained carbon dioxide solution. The device is equipped with an autosampler that limits the necessity to introduce the sample every time.

The instruments enables:• water purity control as per American, Japanese and European pharmacopoeias• confirming the cleanness of technological processes in pharmaceutics

Molecule size analysisMastersizer 3000 (Malvern) and DynaProNanoStar (Wyatt) detectors with a molecule size measuring range from 0.3 nm -3500 µm dispersed both in liquid and in gas.

The instruments allow for:• working with microliter volumes of the solution • analysis of molecules dispersed both in a solvent and in a solid phase


ANALYSIS OF STRUCTURE AND FORMULATION OF MEDICATIONS

The structural analysis allows for determining spatial and constitutional form of chemical compounds. This can be achieved using a few complementary techniques, including nuclear magnetic resonance, VIS, UV and IR spectroscopy, as well as mass spectroscopy.

The Jagiellonian Center of Innovation offers measurements using the above mentioned techniques, with high-quality instruments.

NMR spectroscopyThe NMR Spectroscopy Laboratory of the Jagiellonian Center of Innovation has Avance III HD 400 MHz (Bruker) spectrometer equipped with gradient probes working in a normal (BBO) and inverse detection (BBI). Both probes allow for measuring 1H NMR spectra and spectra of other nuclei (wide-range probes).

The instruments enable:• identification of chemical substances along with the determination of their chemical structure, and often also spatial

structure and compound purity• measurement in the temperature range from -150ºC to + 150ºC• measurement of 1D and 2D NMR spectra, including specialised correlation measurements (NOE, DEPT, COSY, NOESY,

HSQC, HMBC)

IR spectroscopyA laboratory in the Jagiellonian Center of Innovation has a FTIRThermo Scientific Nicolet iS10 spectrometer equipped with ATR attachment. The measurements can be performed in two modes: transmission (KBr tablets or in nujol) and ATR (Attenuated Total Reflection) mode.

The instruments allow for:• verification of active substances and excipients in pharmaceuticals • identification of polymers, co-polymers and additions in plastics, as well as analysis of volatile liquids• measurements without initial preparation of the samples (ATR mode)• measurement of both solid and liquid form

HRMS spectroscopy

A laboratory of the Jagiellonian Center of Innovation has a QTOFImpact II (Bruker) mass spectrometer offering the option of direct injection as well as connection with a liquid uHPLC chromatograph which makes it possible to separate the analyzed mixture. In the instrument, the following ion sources can be used: ESI for large molecular weight compounds (e.g. proteins) and APCIfor analysis of polar and moderately polar compounds of small molecular weight (e.g. alkaloids).

The instruments enable:• broad range of mass analyses (two ionization sources – ESI, APCI)• detailedfragmentation analysis (MS/MS double quadrupole)• mass detection range from 20 to 15000m/z with resolution 10-6 and sensitivity 0.1 ng/mL.

UV-VIS spectroscopyA laboratory of the Jagiellonian Center of Innovation has a UV-VIS spectrophotometer (Agilent Technologies Cary 60).

UV-VIS measurements allow for:• monitoring of chemical and biological reactions kinetics• recording absorption spectra• measuring absorbance profile in the range from 190 nm to 1100 nm• assessment of raw material as well as end-product quality• analysis of photochemical reaction in-situ during light exposure of the sample

The instrument is equipped with anoptical fiber probe for direct measurement of absorbance in a container different than a cuvette.

Analysis of medication formulationThe Jagiellonian Center of Innovation performs pharmaceutical analyses related to the monitoring of formulation methods for the designed as well as ready medications. A laboratory of the Jagiellonian Center of Innovation has automated instruments as per pharmacopoeia recommendations, which enable the measurement of:• release time of active substance from solid and lipophilic formulations• disintegration time of tablets, suppositories and globules• total deformation time for lipophilic suppositories• crush resistance (with simultaneous monitoring of thickness and diameter) • abrasion resistance

Stability tests and tests of conformance with packaging can be also performed using stability chambers. Further qualitative and quantitative measurements are also possible since the laboratory is equipped with complex analytical instruments.


SURFACE ANALYSIS

In the laboratories of the Jagiellonian Center of Innovation, we offer a broad range of surface analysis services based mainly on Raman spectroscopy and electron microscopy.

Laboratory of Raman SpectroscopyThe laboratory is equipped with high class Raman spectrometer (Witec alpha 300 RSA+) with a confocal microscope, an atomic force microscope (AFM) and a polipherometer. All measurements are performed on the same measurement table, hence transfer of the sample is not necessary.

The instrument allows for:• imaging of diffusion of chosen substances in tablets• measuring homogeneity of solid formulations• analysis of sample topography and confocal measurements• chemical compound testing to allow for the differentiation between their polymorphic forms• studying permeability of semi-liquid formulations, depending on the time and conditions of exposition

Laboratory of Scanning Electron MicroscopyThe scanning electron microscope (Mira3-FEG-SEM, Tescan) with field emission (Schottky emitter) equipped with X-ray energy dispersion spectrometerEDX (Oxford Instruments). The microscope enables working in high, low and variable vacuum.

The microscope is equipped with:• secondary electron detector for high vacuum experiments (Everhart-Thornley detector)• secondary electron detector for low vacuum experiments LVSTD (7-500 Pa)• back-scattered electron detector (BSE)for high and low vacuum experiments• cooling table (Peltier) working in the temperature range from -30°C

The laboratory can study conductive materials (metals and alloys), non-conducting samples, such as ceramics, polymers as well as composite and organic materials.

The instrument allows for:• imaging of conducting and non-conductive samples in high and variable vacuum• research on the morphology and chemical composition of non-conductive samples in low and variable vacuum• qualitative and quantitative analysis of elements with atomic number Z≥5 – surface, point, linear analyses, as well

as maps of element distribution in microareas, with the use of EDX spectrometer • studying biological samples in situ, observation of dynamic changes, such as sample hydratation and dehydratation;

observation of phase transitions is also possible (Peltier’s table)• observation of interactions between the sample and the changing environment


Microbiological analysisIn the laboratories of the Jagiellonian Center of Innovation, microbiological analyses of biological, pharmaceutical and environmental materials are conducted. The diagnostics is performed using a conventional method (smear on a substrate, culture, microscopic analysis) and a new diagnostic method using MALDI-TOF mass spectrometry.

Staining and microscopic assessment of specimens:• automatic specimen staining using Gram’s method: automated

staining instrument PreviColour (bioMerieux)• microscopic assessment of microbiological specimens: microscopic

microorganism identification, assessment of the degree of material purity using a simple biological microscope Eclipse Ni-U with a camera and a video camcorder (Nikon)

• smear test: automatic dilution series 5x1/10 and automatic smear on a Petri dish, with a countable range from 30 to cfu/mL.: Spiral Dilute instrument (Interscience)

• automatic counting of colonies and growth inhibition zones as per EUCAST (European Committee on Antimicrobial Susceptibility Testing), along with statistical analysis of the results: Scan 1200 automatic HD colony Counter (Interscience)

• microscopic photographs of the grown microorganisms: microphotography stand (Kaiser)

Identification of microorganisms using a MALDI TOF mass spectrometeridentification of bacteria, yeast-like fungi, filamentous fungi and mold fungi performed using MALDI TOF mass spectrometry with the use of matrix-assisted laser desorption/ionization with time-of-flight analyzer: MALDI TOF mass spectrometer, Microflex LT, Maldi Biotyper (Bruker).

Identification of a microorganism is performed over a few minutes, as compared to 24–48 hours necessary to identify the microorganism using conventional methods. A protein set, the so-called “molecular fingerprint” of the microorganism is compared to protein profiles of reference strain, thus allowing for the identification of the studied strain. A single colony is sufficient to perform the study.

MEDICAL DIAGNOSTICS


Cell cultures Cell cultures are performed in the Laboratory for Tissue and Cell Culture of the Jagiellonian Center of Innovation, in cleanrooms, as per GMP standards. The rooms have been adapted for culturing human tissues and cells for further clinical use.

Constant monitoring of room parameters, as well as laboratory instruments, while maintaining sterile working conditions guarantees safe work with materials of human origin, with respect to both the biological material prepared to be used as graft and personnel safety. This enables treating patients using the state-of-the-art techniques and the most effective treatment methods, including transplant of autologous cells grown in vitro.

The Tissue Bank allows for material storage to ensure a necessary amount of the material required for transplant in the case when the procedure must be repeated.

The Laboratory for Tissue and Cell Culture offers cooperation within the scope of: • isolation and culture of stem cells from heterogeneous population, including those from bone marrow,

umbilical cord blood, Wharton’s jelly, mobilized peripheral blood and other mature tissues• preparation of the obtained cells for autologous application• cell banking and storage

Cell testsThe cell tests are performed with the use of eukaryotic cells. The most standard methods analyze the cytotoxicity of external, physical and chemical factors towards the cell biology. To obtain this goal, the following tests are performed: MTT, MTS and LDH after the selection of an appropriate cell model and based on the recommended standards.

MTT and MTS tests allow for the measurement of mitochondrial enzyme activity in a cell. Mitochondrial oxidative activity is proportional to the amount of yellow tetrazole MTT or MTS salt reduced to blue formazan (blue coloring can be measured spectrophotometrically). The difference between the tests is as follows: use of MTT, in contrast to MTS, results in an insoluble product that must be suspended in an organic solvent. In strictly defined experimental conditions, these tests may be used to determine the viability of non-dividing but metabolically active cells.

Lactate dehydrogenase (LDH) is an intracellular enzyme. Mechanical damage to a plasma membrane and cell death caused by external activity result in LDHrelease from the cells to the environment. Assaying LDH activity in the supernatant provides the measurement of toxicity of the studied substance against the cells in the culture. Enzymatic reactions used in the described method occur in two steps, finally resulting in a colored product that is assayed spectrophotometrically.

TISSUE ENGINEERING AND CELL TESTS

Contact: Agnieszka Witulska Quality Assurance & Control Manager/Responsible Person K: +48 502 519 629 E: [email protected] You are also invited to cooperate with us in the scope of funds acquisition and research grant projects.

laboratories and research services€¦ · nuclear magnetic resonance, vis, uv and ir spectroscopy,...

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