Recently, spheroids have attracted the wide attention of researchers as 3D models for drug testing. One of the key features of spheroids is that they can be composed of one or more cell types. There is a hypothesis that the use of heterospheroids from several cell types when testing anticancer drugs can better reproduce the structure of the tumor and, as a result, increase the predictive value of the study. To verify this hypothesis, we tested the activity of eight antitumor drugs on homospheroids consisting of PANC-1 pancreatic cancer cells and heterospheroids from three types of cells: PANC-1 pancreatic cancer cells, primary human fibroblasts, and human umbilical vein endothelial cells. It has been found that the use of heterospheroids from several cell types, which more accurately reflect the tumor microenvironment, does not lead to a noticeable change in the activity of drugs. Subcutaneous transplantation was carried out of spheroids from PANC-1 to immunodeficient mice which showed that the obtained model of pancreatic cancer more effectively reproduces the aggressive-tumor phenotype compared to transplantation of cells in suspension.

It is becoming approved that screening of compounds on a platform applying two-dimensionally (2D)-cultured cell lines is incapable to precisely predict clinical activity of drugs; therefore three-dimensional (3D)-culture systems are emerging and show potential for better simulating the physiological cell microenvironment. In this regard, a 3D models that reflect all types of intercellular interactions, as well as the interaction of cells with the extracellular matrix, are becoming increasingly accepted. Spheroids represent one of these models. The purpose of our study was to reveal the antiproliferative and cytotoxic activity of cisplatin, doxorubicin, fluorouracil, and etoposide on Vero CCL-81 and HEK293 cell lines under 2D (monolayer) and 3D (spheroids) culture conditions. The comparison of antiproliferative activity (IC 50 ) and cytotoxic activity (CC 50 ) obtained in different culture conditions revealed that the antiproliferative activity of compounds was higher in 2D while cytotoxic effect was more prominent in 3D. We suggest that cells in 3D are cultured in more physiological conditions, consequently they are more resistant to antiproliferative activity of compounds. More prominent cytotoxic effect in 3D allowed us to theorize that implementation of 3D spheroids-based model will allow to recognize early toxicity at the initial steps of preclinical studies in vitro. Our data approved that in the course of determination of compounds specific activity, cell type, the cell proliferation rate and in the optimal diameter of spheroids for each cell type should be taken into account.

The aim of the study is to investigate chondrospheres surface morphology after cytochalasin D treatment. Primary sheep chondrocytes from passage 3 were used for chondrospheres fabrication. The initial concentration was 8000 cells per spheroid. Chondrospheres diameters, number of microvilli and rounded cells on chondrospheres surface were calculated. Chondrospheres diameters examination was performed using inverted light microscope equipped with digital camera. Microphotographs from scanning electron microscopy were used to estimate the number of microvilli and rounded cells. Morphometric analysis showed that chondrospheres diameter increases and chondrocytes become rounded while the densely packed microvilli remain intact in the majority of chondrocytes after cytochalasin D treatment. However, a previously undescribed phenomenon of progressive microvilli depopulation is observed in a small part of cells. Thus, cytochalasin D significantly affects chondrospheres size and surface morphology.

A method is proposed of fabricating tubular constructs from tissue spheroids (conglomerates of cells up to 200 μm in size) in a nutrient fluid using the acoustic radiation force. The source of the acoustic field is a hollow piezoceramic cylinder with a resonant frequency of 800 kHz. Keeping the obtained structure at 37°C for 24 h fuses the spheroids into a solid tubular viable tissue construct.

We present a 3D study of nanostructural features of a bioprinted tissue spheroid interacting with polyurethane dual-scale biocompatible scaffold manufactured by three-dimensional printing and electrospinning. Three-dimensional analysis of fibroblasts interacting with electrospun polyurethane fibers was conducted using scanning probe nanotomography with an experimental setup combining ultramicrotome and a scanning probe microscope. Three-dimensional reconstruction demonstrates direct visualization of cell membrane protrusions and coherent cell-fiber interfaces, the formation of which is a prerequisite for an efficient tissue engineered implant. Analysis of obtained 3D data allows for quantitative calculation of the important morphological parameters of adhered cells, scaffolds, and cell-scaffold interfaces. The proposed method may be successfully applied to investigate 3D cell-scaffold constructs at nanoscale.

клиническая и экспериментальная морфология / CliniCal and ExpErimEntal morphology 2

This paper presents a study of nanostructural features of contacts of bioprinted tissue spheroids with polyurethane dual scale biocompatible scaffold made by three-dimensional printing and electrospinning. Analysis of nanostructural features of cell contacts was carryed out by scanning probe microscopy with use of experimental setup combining ultramicrotome and scanning probe microscope. Measured mean cell volume is 460 ± 104 μm3, mean contact area of cells with scaffold fibers–104.8 μm2 per cell (16.7% of total cell area). Maximum distance of migrating cells from spheroid border at 48 h. is ~200 μm, what corresponds to mean velocity of cell migration more than 4 μm/h. Obtained quantitative characteristics of micro- and nanostructure of human fibroblast cell contacts with elecrospun polyurethane scaffold secure high efficacy of tissue regeneration with its usage for implanted bioprinted dual scale tissue-engineered scaffolds.

Tissue spheroids formed from fibroblasts using a micromolded non-adhesive hydrogel were located using a three-dimensional (3D) bioprinter on the surface of a nanofibrous polyurethane matrix produced by electrospinning. It was shown that the tissue spheroids attach to the matrix surface within a few hours and completely flatten after several days, indicating high biocompatibility of the matrix used. Tissue structures formed by the attachment and spreading of tissue spheroids on an electrospun matrix are a new technological platform for biofabrication and 3D bioprinting of tissues and organs.

The molecular mechanisms of DNA recognition and modification by EcoRII DNA methyltransferase (M.EcoRII) were studied using 14-mer substrate analogs containing 2-aminopurine or 1′,2′-dideoxy-D-ribofuranose in the M.EcoRII recognition site. The efficiency of DNA binding and methylation depended on the position of a modified nucleoside residue in the recognition site. A structural model of M.EcoRII in complex with substrate DNA and the cofactor analog S-adenosyl-L-homocysteine (AdoHcy) was constructed using the available crystal structures of M.Hha and M.HaeIII and the recent Frankenstein’s monster approach. The amino acid residues interacting with DNA were predicted based on the model. In addition, theoretical and experimental findings made it possible to predict the groups of atoms of the heterocyclic bases of the M.EcoRII recognition site that are presumably involved in the interactions with the enzyme.

Prokaryotic DNA methyltransferase M.SssI recognizes and methylates C5 position of the cytosine residue within the CG dinucleotides in DNA. It is an excellent model for studying the mechanism of interaction between CG-specific eukaryotic methyltransferases and DNA. We have built a structural model of M.SssI in complex with the substrate DNA and its analogues as well as the cofactor analogue S-adenosyl-L-homocysteine (AdoHcy) using the previously solved structures of M.HhaI and M.HaeIII as templates. The model was constructed according to the recently developed "FRankenstein's monster" approach. Based on the model, amino acid residues taking part in cofactor binding, target recognition and catalysis were predicted. We also modeled covalent modification of the DNA substrate and studied its influence on protein-DNA interactions.

EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5'.CC*T/AGG.3' and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2'-deoxyuridine (i(5)dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2'-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i(5)dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly(268)-Met(391) region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.

Affinity modification of EcoRII DNA methyltransferase (M x EcoRII) by DNA duplexes containing oxidized 2'-O-beta-D-ribofuranosylcytidine (Crib*) or 1-(beta-D-galactopyranosyl)thymine (Tgal*) residues was performed. Cross-linking yields do not change irrespective of whether active Crib* replaces an outer or an inner (target) deoxycytidine within the EcoRII recognition site. Chemical hydrolysis of M x EcoRII in the covalent cross-linked complex with the Tgal*-substituted DNA indicates the region Gly268-Met391 of the methylase that is likely to interact with the DNA sugar-phosphate backbone. Both specific and non-specific DNA interact with the same M x EcoRII region. Our results support the theoretically predicted DNA binding region of M x EcoRII.