Research

Cell viability assays

XTT Cell Viability Assay – a colorimetric assay that detects the cellular metabolic activities. During the assay, the yellow tetrazolium salt XTT is reduced to a highly colored formazan dye by dehydrogenase enzymes in metabolically active cells. This extracellular reduction is carried out by electron transport across the plasma membrane of a living cell. The amount of XTT reduced is reflective of the cellular metabolic activity. The absorbance can be measured and compared to the absorbance of a control solution of untreated cells to determine if cellular metabolic activity has increased or decreased. Thus, XTT can be used to assess cell proliferation or cytotoxicity of drugs. The formazan dye can be quantified by measuring the absorbance at wavelength 490 nm using a plate-reading spectrophotometer (BioTek).

Example

(A) viability of mouse embryonic fibroblasts NIH3T3, incubated with SPIONs for 3, 24 and 48 h. Toxicity of nanoparticles was investigated using XTT cell viability assay; (B) XTT plate images of NIH3T3 cells incubated with 32.5 ng/mL of SPION nanoparticles for 0, 24 and 48 h. (Jarockyte et al, 2016)

LDH Cytotoxicity Assay – a colorimetric method for quantifying cellular cytotoxicity assays. Lactate dehydrogenase (LDH) is a cytosolic enzyme present in many different cell types. Plasma membrane damage releases LDH into the cell culture media. Extracellular LDH in the media can be quantified by a coupled enzymatic reaction in which LDH catalyzes the conversion of lactate to pyruvate via NAD⁺ reduction to NADH. Diaphorase then uses NADH to reduce a tetrazolium salt (INT) to a red formazan product that can be measured at 490 nm using a plate-reading spectrophotometer (BioTek). The level of formazan formation is directly proportional to the amount of LDH released into the medium, which is indicative of cytotoxicity.

Example

Viability of MDA-MB-231 and MCF-7 breast cancer cells incubated at different concentrations of dNPs and dNPs-Ce₆ complex for 24 h. No cytotoxicity was observed for MCF-7 cells, whereas the MDA-MB-231 cells viability decreased to 95% with a p ≤ 0.05 significance (indicated by an asterisk). (Skripka et al., 2019)

Determination of cells viability using the Adam-MC Automatic Cell Counter (Digital Bio). The nucleus of total and only non-viable cells are stained with Accustain solutions. AccuStain Solution T for the total cell counting is composed of the fluorescent dye Propidium Iodide (PI) and lysis solution. AccuStain Solution N for the non-viable cell counting is composed of the PI and PBS. PI does not enter cells with intact cell membranes or active metabolism. As a result, the nuclei of non-viable cells will only be stained. The viability is automatically calculated by the ADAM-MC software after each measurement of the total cells and the non-viable cells.

Example

Cell viability of MCF-7 and MDA-MB-231 cells incubated with BSA-Au NCs (56 mg/mL), BSA, Au-MES NCs (45 mg/mL), and MES solutions. Error bars show the standard deviations. * indicates significant differences compared to the non-treated cells (Control) (p ≤ 0.05); # indicates significant differences between the MCF-7 and MDA-MB-231 cell lines (p ≤ 0.05). (Matulionyte et al., 2017).

Flow cytometry is laser-based technology used for the analysis of cell characteristics. It allows simultaneous multi-parameter analysis of single cells. Single-cell suspension ensures that every cell is analyzed independently. Due to speed, sensitivity and multiple parameters analyzed at the same time, flow cytometry provides the statistical power to analyze and characterize millions of cells. Flow cytometry is used to:

• detect the expression of cell surface (various CD (cluster of differentiation) molecules (e.g. CD44, CD24, CD90, CD34, etc.)) and intracellular proteins (e.g. Ki-67);
• assess cell viability (Annexin V, PI (propidium iodide));
• assess generation of reactive oxygen species (ROS) (CellROX);
• accumulation dynamics of various fluorescent nanoparticles (quantum dots, gold nanoparticles) and dyes (rhodamine 6G, photosensitizers);
• characterization of different cell types in a heterogeneous cell population (by the immunophenotype or morphological differences, for example, white blood cells).

Example:

Left – Apoptosis analysis of MDA-MB-231 cells after incubation with gold nanoclusters (Au NCs) for 24 and 48 h by flow cytometry, using Annexin V/ PI apoptosis assay. Right – Accumulation of photoluminescent gold nanoclusters (Au NCs) in MCF-7 and MDA-MB-231 cells after 3, 6, and 24 h of incubation represented as mean PL intensity of per cell. Control represents autofluorescence of non-treated cells. (Matulionyte et al., 2017).

3D spheroid cell cultures

3D spheroid cell culture – self-assembled clusters of cell colonies cultured in environments where cell-cell interactions dominate over cell-substrate interactions, and they naturally mimic avascular tumors with inherent oxygen and nutrient gradients. 3D spheroid cell culture could be used as avascular tumor model to investigate the uptake and distribution of nanoparticles in the tumor cells.

Schematic picture of 3D spheroid cell culture formation process. (Jarockyte et al, 2018 )

In our laboratory, 3D spheroid cell cultures are formed using the hanging drop technique. Cells suspension is poured in hanging drop plate. A drop with dispersed cells is formed. In this drop, cells are making cell-to-cell contacts and aggregate until spheroid is formed. Diameters of spheroids depend on the cell line, initial number of cells and growth time.

Cultivation of 3D spheroid cell cultures in hanging drop plates.

Diameters of spheroids depend on the cell line, initial number of cells and growth time. Also spheroids could vary in cell density, depending on which cell line is used. Example of spheroid formation in hanging drop plate is showed below. 

NIH3T3 cells spheroid formation process in hanging drop plate. Cell images were taken with Nikon Eclipse Te2000-S microscope bright-field mode at different stages of formation. At the beginning, dispersed cells (50 cells/μL) were distributed in the hanging drop. In the drop, cells made cell-to-cell contacts and aggregated until a spheroid was formed. (Jarockyte et al, 2018 )

The rapidly evolving nanotechnology in the field of cancer research requires a better understanding of nanoparticle distribution in the tissue and uptake in solid tumors. Thus we use 3D cell cultures as in vitro models of native complex tumor microenvironment for primary nanoparticles testing.

Accumulation of QDs (red color) in fully formed NIH3T3 spheroid (50 cells/μL, grown for 72 h before QDs were added, incubated for 24 h with QDs) (left) and QD accumulation in NIH3T3 spheroid which was formed from the cells, pre-incubated with QDs (50 cells/μL, grown for 96 h in spheroid) (right). Cell nuclei stained with nucleus stain Hoechst (blue color) (λ_ex= 404 nm). (Jarockyte et al, 2018 )

3D cell cultures could be used not only for investigation of nanoparticles distribution in tissue model, but also to test nanoparticles potential in therapy. For example, using 3D cell cultures we could test if nanoparticles are effective in photodynamic therapy.

After 24 h incubation with dNPs or dNPs-Ce₆, spheroids were irradiated either by 806 or 980 nm light (0.5 W cm⁻²; 900 J cm⁻²) and treated with live/dead cell viability markers (green—alive, red—dead). Only the dNPs-Ce₆ complex under 980 nm light induced damage to MDA-MB-231 spheroid, while none was observed when the same complex was excited by the therapy independent 806 nm light. Scale bars in all images correspond to 200 μm. (Skripka et al., 2019)

Optical biopsy

Optical methods (optical biopsy) carry information about intrinsic properties of the tissue and provide unique possibility to study the objects quickly and non-invasively.

The method can be applied for the identification of the diseased tissues, such as cancer or for the differentiation between tissues otherwise being visually indiscernible, such as conduction system of the heart.

Instruments:

Edinburgh FLS 920:

• double grating monochromators,
• heated or cooled cuvette and solid sample holders,
• fiber optics supplement;
• Xe discharge lamp;
• pulsed (ns) nitrogen lamp;
• pulsed diodes (405 nm...),
• 980 nm CW laser.
• ultra sensitive UV-VIS detector,
• IR detector and
• ultra fast UV-VIS detector for lifetime measurements.

Experiments that could be performed:

Steady state measurements;

Steady state measurements is a simple method to investigate/characterize objects based on their general composition and depends mostly on the present fluorophores. 

• Ex vivo samples measurements;

Fluorescence spectra of HCS, CT and MC tissues under 330 nm and 380 nm excitation. A vertical line marks 460 nm (JBO,16(10), 107001 (October 2011).

• Solutions measurements;
• Nanoparticles (nanomaterials) measurements;
• Upconversion nanoparticles measuremnts;
• Cell suspensions measurements.

Kinetic (lifetime) measurements;

Fluorescence lifetime is the characteristic average time that a fluorophore spends in the excited state. It varies with the molecular environment (including pH, ion concentration and binding, enzymatic activity, temperature) but usually does not depend on fluorophore concentration, thus, environmental effects and fluorophores with overlapping spectra could be distinguished. 

• Ex vivo samples measurements;
• Solutions measurements;
• Nanoparticles (nanomaterials) measurements;
• Upconversion nanoparticles measuremnts;
• Cell suspensions measurements.

Fluorescence intensity decay registered at 482 nm. MC (picture A), HCS (picture B) and CT (picture C). (10.3952/lithjphys.51415).

Scanning of ex vivo samples;

The precise spectroscopic mapping of the tissue samples allows true spectral imaging with the spectral resolution of a 0,01 nm and single photon counting sensitivity.

Planar scanning (mapping) of the specimen. Picture in the daylight (left panel); spectral image (middle panel) - each pixel represents the ratio Rtiss(460) = Iex330/Iex380; Combined picture of spectral and fluorescence images (right panel) (JBO,16(10), 107001 (October 2011)).

In vivo measurements (using fiber optics probe)

The possibility to perform spectral measurements from live objects makes optical biopsy very attractive method for early diagnosis and for decision making during the surgery procedures.

The simplified equipment makes it possible to implement it in any type of the procedures.

• Oropharyngeal diagnostics;

(Intra-arterial administration of photosensitizer and fluorescence detection of  tumorous site.)

• Cervical neoplasia detection;

(Fluorescence spectra from neoplastic, healthy cervical tissue and skin after topical ALA application in the same patient (BMC Women's Health (2015) 15:35)

• Experimental animal measurements;