Cells In Explants Research Articles

NMOSD IgG Impact Retinal Cells in Murine Retinal Explants.

Neuromyelitis optica spectrum disorders (NMOSD) are chronic inflammatory diseases of the central nervous system, characterized by autoantibodies against aquaporin-4. The symptoms primarily involve severe optic neuritis and longitudinally extensive transverse myelitis. Although the disease progression is typically relapse-dependent, recent studies revealed retinal neuroaxonal degeneration unrelated to relapse activity, potentially due to anti-aquaporin-4-positive antibodies interacting with retinal glial cells such as Müller cells. In this exploratory study, we analysed the response of mouse retinal explants to NMOSD immunoglobulins (IgG). Mouse retinal explants were treated with purified IgG from patient or control sera for one and three days. We characterized tissue response patterns through morphological changes, chemokine secretion, and complement expression. Mouse retinal explants exhibited a basic proinflammatory response ex vivo, modified by IgG addition. NMOSD IgG, unlike control IgG, increased gliosis and decreased chemokine release (CCL2, CCL3, CCL4, and CXCL-10). Complement component expression by retinal cells remained unaltered by either IgG fraction. We conclude that human NMOSD IgG can possibly bind in the mouse retina, altering the local cellular environment. This intraretinal stress may contribute to retinal degeneration independent of relapse activity in NMOSD, suggesting a primary retinopathy.

Abstract P104: EXAMINING THE EFFECTS OF FOLLISTATIN ON CONTRACTION OF VESSELS FROM NORMOTENSIVE AND HYPERTENSIVE RATS

The lab has previously shown that follistatin (FST), a glycoprotein inhibitor of activins, reduced blood pressure in the Spontaneously Hypertensive Rat (SHR) model of Essential Hypertension. Short-term exposure (30 minutes) to FST also reduced vessel contraction induced by potassium (KCl). This study aims to illustrate the mechanisms through which FST inhibits potassium-induced contraction in SHR and normotensive (WKY) rats. Function of SHR or WKY resistance mesenteric arteries was tested in response to KCl and ion channel modulators using wire myography. Cultures of primary smooth muscle cells (SMC) were also established from these vessels using cell explant technique. The calcium sensor dye Fluo-4 AM was used to examine calcium influx on a cellular level. To test whether FST effects were due to activin neutralization or activin-receptor inhibition, neutralizing antibodies against activin A and B or an activin receptor inhibitor (SB 431542) were used, respectively. Only activin A neutralization in the SHR reduced KCl-induced contraction similarly to FST. Activin receptor inhibition also reduced KCl constriction similarly to FST. When directly stimulated with Activin A, both WKY and SHR vessels displayed augmented KCl contraction compared to controls but the effect was more pronounced in the SHR. Preliminary Fluo-4 results showed that FST decreased KCl-induced calcium influx in SMC. Since activin A was shown to increase L-type voltage-gated Ca2+channel activity, an agonist (FPL 64176) of this channel was used to induce contraction. Although FST reduced KCl-induced contraction, it did not affect FPL-induced contraction in either SHR or WKY. Thus, Activin A neutralization reproduces FST reduction of KCl-induced vessel contraction. FST did not reduce FPL-induced contraction, suggesting that its inhibition of KCl-induced contraction is not through effects on this channel but rather an alternate Ca2+ or K-ATP channel.

Cinchonine and cinchonidine alleviate cisplatin-induced ototoxicity by regulating PI3K-AKT signaling.

Cinchonine (CN) and its isomer cinchonidine (CD), two of the common cinchona alkaloids, are wildly used as antimalarial drugs. However, the effects of CN and CD on the auditory system are unknown. Molecular docking and molecular dynamics (MD) simulation were used for predicting effective drugs. The CCK-8 assay was conducted for assessing cell viability in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells. MitoSox Red staining revealed reactive oxygen species (ROS) amounts. TMRM staining was used to assess the mitochondrial membrane potential (ΔΨm). Immunofluorescence staining of myosin 7a was used to examine hair cells (HCs) in cisplatin-treated neonatal mouse cochlear explants, while TUJ-1 immunostaining was used for the detection of spiral ganglion neurons (SGNs). Cleaved caspase-3 and TUNEL immunostaining were utilized for apoptosis assessment. Immunoblot was carried out to detect PI3K-AKT signaling effectors. Pretreatment with CN or CD significantly increased cell viability and reduced mitochondrial dysfunction and ROS accumulation in cisplatin-treated HEI-OC1 cells. Immunofluorescent staining of cochlear explants showed that CN and CD attenuated cisplatin-induced damage to SGNs and HCs. Immunoblot revealed that CN and CD downregulated the expression of cleaved caspase-3 and activated PI3K-AKT signaling in cisplatin-injured HEI-OC1 cells. CD and CN can reduce ototoxicity caused by cisplatin and might help treat cisplatin-associated hearing loss.

Susceptibility of perivenous macrophages to PRRSV-1 subtype 1 LV and PRRSV-1 subtype 3 Lena using a new vein explant model.

Vessel pathology such as increased permeability and blue discoloration is frequently observed with highly pathogenic PRRSV strains. However, data concerning the viral replication in the environment of blood vessels are absent. In the present study, ex vivo models with swine ear and hind leg vein explants were established to study the interaction of PRRSV-1 subtype 1 reference strain LV and highly pathogenic subtype 3 strain Lena with perivenous macrophages. The replication characteristics of these two strains were compared in vein explants by immunofluorescence analysis. The explants maintained a good viability during 48 hours of in vitro culture. We found that CD163-positive macrophages were mainly present around the veins and their number gradually decreased with increasing distance from the veins and longer incubation time. More CD163+Sn- cells than CD163+Sn+ cells (6.6 times more) were observed in the vein explants. The Lena strain demonstrated a higher replication level than the LV strain, with approximately 1.4-fold more infected cells in the surrounding areas of the ear vein and 1.1-fold more infected cells in the leg vein explants at 48 hours post inoculation. In both LV and Lena inoculated vein explants, most infected cells were identified as CD163+Sn+ (> 94%). In this study, an ex vivo vein model was successfully established, and our findings will contribute to a better understanding of the vein pathology during viral infections (e.g., PRRS, classical and African swine fever).

Optimization of in vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation of soybean for visual screening of transformants using RUBY.

In vitro and ex vitro Agrobacterium rhizogenes-mediated hairy root transformation (HRT) assays are key components of the plant biotechnology and functional genomics toolkit. In this report, both in vitro and ex vitro HRT were optimized in soybean using the RUBY reporter. Different parameters including A. rhizogenes strain, optical density of the bacterial cell culture (OD600), co-cultivation media, soybean genotype, explant age, and acetosyringone addition and concentration were evaluated. Overall, the in vitro assay was more efficient than the ex vitro assay in terms of the percentage of induction of hairy roots and transformed roots (expressing RUBY). Nonetheless, the ex vitro technique was deemed faster and a less complicated approach. The highest transformation of RUBY was observed on 7-d-old cotyledons of cv. Bert inoculated for 30 minutes with the R1000 resuspended in ¼ B5 medium to OD600 (0.3) and 150 µM of acetosyringone. The parameters of this assay also led to the highest percentage of RUBY through two-step ex vitro hairy root transformation. Finally, using machine learning-based modeling, optimal protocols for both assays were further defined. This study establishes efficient and reliable hairy root transformation protocols applicable for functional studies in soybean.

3D printed cathodes for implantable abiotic biofuel cells

3D printing has recently triggered huge attention in several fields such as construction, artificial tissue engineering, food fabrication, wearable electronics, and electrochemical energy storage.This work investigates the fabrication of a 3D-printed abiotic cathode for implantable glucose/oxygen biofuel cells. The ink formulation was optimized to get printable ink with high electro-catalytic activity. Electrode macro porosity was screened in order to identify the better compromise between electrode density and electrochemical performance. A maximum current density of 260 μA/cm2 was obtained with cylindrical electrodes with linear mesh infill and a volumic infill rate of 40%.A complete biofuel cell was assembled using a 3D-printed abiotic cathode and an enzymatic anode in the form of a compressed pellet showing maximum power and current densities of 80 μW/cm2 and 320 μA/cm2, respectively. Moreover, the hybrid biofuel cell was implanted in the intraabdominal region of a rat for three months and after cell explantation, the abiotic cathode displayed a 50% decrease in the current density while the enzymatic anode did not display any residual activity.The 3D printed electrode displayed a 2–3.6 fold increase in current density when compared to homolog 2D electrodes.

The Impact of Gold Nanoparticles on Somatic Embryogenesis Using the Example of Arabidopsis thaliana.

Although the influence of nanoparticles (NPs) on developmental processes is better understood, little is known about their impact on somatic embryogenesis (SE). This process involves changes in the direction of cell differentiation. Thus, studying the effect of NPs on SE is essential to reveal their impact on cell fate. This study aimed to examine the influence of gold nanoparticles (Au NPs) with different surface charges on the SE of 35S:BBM Arabidopsis thaliana, with particular emphasis on the spatiotemporal localization of pectic arabinogalactan proteins (AGPs) and extensin epitopes in cells changing the direction of their differentiation. The results show that under the influence of nanoparticles, the explant cells of 35S:BBM Arabidopsis thaliana seedling origin did not enter the path of SE. Bulges and the formation of organ-like structures were observed in these explants, in contrast to the control, where somatic embryos developed. Additionally, spatiotemporal changes in the chemical composition of the cell walls during the culture were observed. Under the influence of Au NPs, the following effects were observed: (1) explant cells did not enter the SE pathway, (2) the impacts of Au NPs with different surface charges on the explants were variable, and (3) the compositions of the analyzed pectic AGPs and extensin epitopes were diverse in the cells with different developmental programs: SE (control) and non-SE (treated with Au NPs).

Indirect regeneration in Ficus lyrata Warb. and metabolite profiles influenced by nitric oxide and Plant growth regulators

BackgroundTo establish an indirect regeneration protocol in Ficus lyrata, a three-phase experiment (callus induction, morphogenic callus induction, and plant regeneration) based on auxin, cytokinin, and nitric oxide interactions was designed and implemented using leaf explants. The metabolite profiles (amino acid profile, total phenolic content, total soluble sugars, and total antioxidant activity) alteration patterns were also investigated to determine the metabolites contributing to the progress of each phase.ResultsResults demonstrated that 11 out of 48 implemented treatments resulted in morphogenic callus induction (morphogenic treatments), and nitric oxide played a key role in increasing efficiency from 13 to 100%. More importantly, nitric oxide cross-talk with cytokinins was necessary for shoot regeneration from morphogenic calli. Only 4 out of all 48 implemented treatments were capable of shoot regeneration (regenerative treatments), and among them, PR42 treatment led to the highest shoot regeneration rate (86%) and maximum mean number of shoot/explant (10.46). Metabolite analyses revealed that the morphogenic and regenerative treatments followed similar metabolite alterations, which were associated with increased biosynthesis of arginine, lysine, methionine, asparagine, glutamine, histidine, threonine, leucine, glycine, serine amino acids, total soluble sugars content, and total antioxidant activity. On the contrary, non-morphogenic and non-regenerative treatments caused the accumulation of a significantly greater total phenolic content and malondialdehyde in the explant cells, which reflexed the stressful condition of the explants.ConclusionsIt could be concluded that the proper interactions of auxin, cytokinins, and nitric oxide could result in metabolite biosynthesis alterations, leading to triggering cell proliferation, morphogenic center formation, and shoot regeneration.

Screening Smoke Tree (Cotinus coggygria Scop.) on Osmotic Stress using Polyethylene Glycol 6000 in vitro

Background: Droughts during the growing season are becoming increasingly common due to climate change and global warming. The study’s goal study was to assess the adaptation mechanisms of C. coggygria Scop. under simulated water deficit conditions using indicators characterized changes in the leaf stomatal apparatus and photosynthesizing system. Methods: The influence of osmotic polyethylene glycol 6000 on the research object C. coggygria regenerants was studied in vitro. Such indicators as morphological changes, pigment content, stomatal density and size and stomatal slit size were quantified. Result: Due to the influence of osmotic stress, there was a pigment content increase in the leaf plate on osmotic media. Stomatal density raised by 27.0-29.8% in explant cells cultivated on PEG 4.0-6.0%. Under the osmotic influence, there was a drop in the stomatal cells’ area and shape and the ratio of stomatal length to width in the regenerant leaves decreased from 1.5 to 1.0-1.1. These results indicate the adaptation of C. coggygria regenerants to stress caused by an artificial drought in vitro. The changes’ identification in the stomatal apparatus and the C. coggygria pigment ratio will accelerate the selective screening of drought-resistant plants for use in protective forestry.Background: Droughts during the growing season are becoming increasingly common due to climate change and global warming. The study’s goal study was to assess the adaptation mechanisms of C. coggygria Scop. under simulated water deficit conditions using indicators characterized changes in the leaf stomatal apparatus and photosynthesizing system.

Ex vivo expansion of primary cells from limb tissue of Pleurodeles waltl.

Pleurodeles waltl is coming to light as a model animal, especially in regeneration studies, but deep studies on the molecular mechanisms have been limited due to the absence of primary tissue cells for wide usage. Therefore, we aimed to grow primary cells from limb tissue of P. waltl for in vitro experiments. Limb tissues were cut into small pieces and seeded as "explants" on culture dishes coated with fibronectin and gelatin. Compared to the control without coating, both fibronectin and gelatin supported quicker outgrowth of cells from explants and faster cell adhesion, and fibronectin showed significantly better performance than gelatin. Interestingly, the doubling time of cells on fibronectin- and gelatin-coated surfaces was almost the same (42.39 ± 2.79 h vs. 42.91 ± 3.69 h) and was not significantly different from that on non-coated plates (49.64 ± 3.63 h). The cryopreserved cells were successfully recovered and showed a multiplication capacity that was similar to that of fresh cells. Senescent cells were barely detected even after long-term sub-culture (>15 passages). Moreover, enhanced fluorescence of MitoSOX™ Red in cells under H2 O2 exposure confirmed the respondence to chemical stimuli. Collectively, our results show that we are able to grow enough good-quality cells from P. waltl limb tissue for in vitro experiments, and fibronectin coating provides the best biocompatible environment for cell outgrowth and attachment.

Nicotinic acid addition to equine frozen semen extender influences the number of sperm bound to bovine oviduct explants

Usually, equine non-capacitated sperm bind to the isthmus region of the oviduct before ovulation occurs, a process believed essential to prepare them to fertilize the oocyte. However, during semen freezing most of the seminal plasma, a rich source of antioxidants is discarded, resulting in a higher sperm vulnerability to oxidative damage. Thus, the aim of this study was to investigate the influence of adding nicotinic acid, an antioxidant, to equine frozen semen extender to improve the binding capacity of frozen equine sperm to bovine oviduct cell explants, as an indirect way to investigate the sperm fertilizing capability. To obtain bovine oviducts, reproductive systems of abattoir slaughtered cows were transported in saline solution at 35°C to the laboratory. After sectioning the uterus-tubal junction ipsilateral to the ovaries without a corpus luteum, the isthmus region was pressed towards the uterus and its contents expelled in a Petri dish with culture medium. The oviduct contents with medium were decanted into a tube and after 5 minutes, disaggregated using a needle and syringe three times. Then, the contents were deposited in Petri dishes and cultured for 24 hours in an incubator with 5% CO2 at 38°C. Semen from ten stallions was frozen in INRA96 (control, no nicotinic acid) and with 10mM and 20mM nicotinic acid. Thawed spermatozoa(30 × 106 sperm/mL) were co-incubated with 20 oviduct explants (aggregated oviduct cells) for 30min in an incubator with 5% CO2 at 38°C. Subsequently, the explants were washed with medium to remove the loosened sperm, placed on a glass slide, and covered with a coverslip. The number of sperm bound per nm of oviduct was observed with phase contrast microscopy (40x) and counted with ImageJ software. For statistical analysis, mean and standard error were calculated. When the data did not show normal distribution, logarithmic transformation was performed and the log mean values were evaluated with variance analysis and compared with Duncan's test. The probability of P<0.05 was considered significant. The addition of 10mM nicotinic acid to equine frozen semen extender increased the number of sperm bound to the explants (79.9 ± 9.5 sperm/nm explant) compared to the control (49.1 ± 6.8 sperm/nm explant, P<0.05) and 20mM nicotinic acid treatment (65.6 ± 8.3 sperm/nm explant, P<0.05). In conclusion, the addition of 10mM nicotinic acid to equine freezing extenders improved the number of sperm bound to bovine oviduct explants. Hence, nicotinic acid may be a useful addition to freezing extenders.

Resolved Psoriasis with Abundant Oleic Acid in Stratum Corneum Exhibits Lower T-Cell–Driven IL-17 Signature

Relapses of psoriasis involve T cells that stem and survive in the skin. Inherited from previous flares, the tissue-resident memory T cells are epidermal IL-17-producing CD8+ and IL-22-producing CD4+ T cells. Because the capacity of resident memory T cells to take in fatty acids is essential for their residence and function, the surface composition of fatty acids may affect underlying T-cell populations. In patients treated with biologics, we used gas chromatography/mass spectrometry to decipher the fatty acid composition in both resolved and nonlesional sites. Skin T cells were activated by OKT-3 in explants from the same body sites to perform bulk transcriptomic analysis (Nanostring). The fatty acid composition differed between skin from healthy donors and normal-looking skin of patients with psoriasis but not further between nonlesional and resolved skin. Patients in whom the resolved skin was rich in oleic acid had lower T-cell-driven IL-17 epidermal transcriptomic signature upon activation of T cells in skin explants. The skin lipid composition is linked with the functions of the underlying epidermal T cells. Testing the modulating effect of custom fatty acids on skin resident T cells could help with coming closer to disease oblivion in inflammatory skin diseases.

Apoplastic and Symplasmic Markers of Somatic Embryogenesis.

Somatic embryogenesis (SE) is a process that scientists have been trying to understand for many years because, on the one hand, it is a manifestation of the totipotency of plant cells, so it enables the study of the mechanisms regulating this process, and, on the other hand, it is an important method of plant propagation. Using SE in basic research and in practice is invaluable. This article describes the latest, but also historical, information on changes in the chemical composition of the cell wall during the transition of cells from the somatic to embryogenic state, and the importance of symplasmic communication during SE. Among wall chemical components, different pectic, AGP, extensin epitopes, and lipid transfer proteins have been discussed as potential apoplastic markers of explant cells during the acquisition of embryogenic competence. The role of symplasmic communication/isolation during SE has also been discussed, paying particular attention to the formation of symplasmic domains within and between cells that carry out different developmental processes. Information about the number and functionality of plasmodesmata (PD) and callose deposition as the main player in symplasmic isolation has also been presented.

Label-free Chemical Characterization of Polarized Immune Cells in vitro and Host Response to Implanted Bio-instructive Polymers in vivo Using 3D OrbiSIMS.

The Three-dimensional OrbiTrap Secondary Ion Mass Spectrometry (3D OrbiSIMS) is a secondary ion mass spectrometry instrument, a combination of a Time of Flight (ToF) instrument with an Orbitrap analyzer. The 3D OrbiSIMS technique is a powerful tool for metabolic profiling in biological samples. This can be achieved at subcellular spatial resolution, high sensitivity, and high mass-resolving power coupled with MS/MS analysis. Characterizing the metabolic signature of macrophage subsets within tissue sections offers great potential to understand the response of the human immune system to implanted biomaterials. Here, we describe a protocol for direct analysis of individual cells after in vitro differentiation of naïve monocytes into M1 and M2 phenotypes using cytokines. As a first step in vivo, we investigate explanted silicon catheter sections as a medical device in a rodent model of foreign body response. Protocols are presented to allow the host response to different immune instructive materials to be compared. The first demonstration of this capability illustrates the great potential of direct cell and tissue section analysis for in situ metabolite profiling to probe functional phenotypes using molecular signatures. Details of the in vitro cell approach, materials, sample preparation, and explant handling are presented, in addition to the data acquisition approaches and the data analysis pipelines required to achieve useful interpretation of these complex spectra. This method is useful for in situ characterization of both in vitro single cells and ex vivo tissue sections. This will aid the understanding of the immune response to medical implants by informing the design of immune-instructive biomaterials with positive interactions. It can also be used to investigate a broad range of other clinically relevant therapeutics and immune dysregulations. Graphical overview.

NFATc1 Is a Central Mediator of EGFR-Induced ARID1A Chromatin Dissociation During Acinar Cell Reprogramming

Loss of AT-rich interactive domain-containing protein 1A (ARID1A) fosters acinar-to-ductal metaplasia (ADM) and pancreatic carcinogenesis by down-regulating transcription programs controlling acinar cell identity. However, how ARID1A reacts to metaplasia-triggering environmental cues remains elusive. Here, we aimed to elucidate the role of ARID1A in controlling ductal pancreatic gene signatures and deciphering hierarchical signaling cues determining ARID1A-dependent chromatin regulation during acinar cell reprogramming. Acinar cell explants with differential ARID1A status were subjected to genome-wide expression analyses. The impact of epidermal growth factor receptor (EGFR) signaling, NFATc1 activity, and ARID1A status on acinar reprogramming processes were characterized by exvivo ADM assays and transgenic mouse models. EGFR-dependent ARID1A chromatin binding was studied by chromatin immunoprecipitation sequencing analysis and cellular fractionation. EGFR signaling interferes with ARID1A-dependent transcription by inducing genome-wide ARID1A displacement, thereby phenocopying ARID1A loss-of-function mutations and inducing a shift toward ADM permissive ductal transcription programs. Moreover, we show that EGFR signaling is required to push ARID1A-deficient acinar cells toward a metaplastic phenotype. Mechanistically, we identified the transcription factor nuclear factor of activated T cells 1 (NFATc1) as the central regulatory hub mediating both EGFR signaling-induced genomic ARID1A displacement and the induction of ADM-promoting gene signatures in the absence of ARID1A. Consequently, pharmacologic inhibition of NFATc1 or its depletion in transgenic mice not only preserves genome-wide ARID1A occupancy, but also attenuates acinar metaplasia led by ARID1A loss. Our data describe an intimate relationship between environmental signaling and chromatin remodeling in orchestrating cell fate decisions in the pancreas, and illustrate how ARID1A loss influences transcriptional regulation in acinar cell reprogramming.

Türk Civanperçemi (Achillea gypsicola Hub. Mor.) Hücre Süspansiyon Kültürlerinde Fenilpropanoid Metabolitlerin Işık Aracılığıyla Biosentezi

Turkish yarrow (Achillea gypsicola Hub. Mor.) contains medicinally valuable bioactive metabolites. Light plays a key role in many physiological events in plants such as the accumulation of phenylpropanoid metabolites, particularly photosynthesis. The aim of this study was to determine the effects of light on the biosynthesis of phenylpropanoid metabolites and biomass growth in Turkish yarrow cell suspension cultures. In vitro plantlets obtained from seeds were used as explant sources and cell cultures of A. gypsicola were established using stem segment-derived callus tissues. Then, 8-day-old cell cultures kept in a B5 medium supplemented with 0.5 mg/L BA (benzylaminopurine)+0.5 mg/L NAA (naphthalene acetic acid) were incubated in light and dark environments. Cells were harvested five times (1st, 2nd, 3rd, 6th, and 12th day) in total. The amount of camphor was determined by gas chromatography-mass spectrometry (GC–MS). Other parameters were calculated using the spectrophotometric method. The total phenolic amount increased depending on the sampling time in dark and light conditions. Phenolic accumulation was increased by 29.25% compared to the initial culture. Also, light application increased the amount of camphor 1.12 times, the amount of flavanol 4.9 times, and the amount of flavonol 0.5 times. These results clearly demonstrate the effect of light on cell growth and phenylpropanoid metabolite accumulation in A. gypsicola cell cultures.

Imaging of lactate metabolism in retinal Müller cells with a FRET nanosensor

Müller cells, the glial cells of the retina, provide metabolic support for photoreceptors and inner retinal neurons, and have been proposed as source of the significant lactate production of this tissue. To better understand the role of lactate in retinal metabolism, we expressed a lactate and a glucose nanosensor in organotypic mouse retinal explants cultured for 14 days, and used FRET imaging in acute vibratome sections of the explants to study metabolite flux in real time. Pharmacological manipulation with specific monocarboxylate transporter (MCT) inhibitors and immunohistochemistry revealed the functional expression of MCT1, MCT2 and MCT4 in Müller cells of retinal explants. The introduction of FRET nanosensors to measure key metabolites at the cellular level may contribute to a better understanding of heretofore poorly understood issues in retinal metabolism.

Experimental in vitro, exvivo and in vivo models in prostate cancer research.

Androgen deprivation therapy has a central role in the treatment of advanced prostate cancer, often causing initial tumour remission before increasing independence from signal transduction mechanisms of the androgen receptor and then eventual disease progression. Novel treatment approaches are urgently needed, but only a fraction of promising drug candidates from the laboratory will eventually reach clinical approval, highlighting the demand for critical assessment of current preclinical models. Such models include standard, genetically modified and patient-derived cell lines, spheroid and organoid culture models, scaffold and hydrogel cultures, tissue slices, tumour xenograft models, patient-derived xenograft and circulating tumour cell eXplant models as well as transgenic and knockout mouse models. These models need to account for inter-patient and intra-patient heterogeneity, the acquisition of primary or secondary resistance, the interaction of tumour cells with their microenvironment, which make crucial contributions to tumour progression and resistance, as well as the effects of the 3D tissue network on drug penetration, bioavailability and efficacy.

COMPARATIVE ANALYSIS OF THE EFFICIENCY OF THE METHODS&#x0D; OF REGENERATION FROM RAPESEED EXPLANTS OF MAGNAT AND&#x0D; LEADER VARIETIES

A comparative analysis of the effectiveness of organogenesis from the callus cells of hypocotyl segment explants in rapeseed varieties Leader and Magnat regionalized in Belarus was carried out. It was established that the Leader, compared to the Magnat variety, is more suitable for organogenesis from plant explants in vitro. It was shown that for the Leader variety the regeneration efficiency is up to 10 plants adapted ex vitro per explant, when using a method, according to which hypocotyl explants are first exposed to auxin (2,4-dichlorophenoxyacetic acid, 1 mg/l), and then to cytokinin (6-benzylaminopurine, 5–8 mg/l).

Does the ovarian surface epithelium differentiate into primordial follicle and primary follicle precursor structures?

Amaç: Ovaryum yüzey epiteli hücrelerinin farklılaşma kapasitelerini hem hücre kültürü şartlarında hem de ovaryum doku kesitlerinde araştırmaktır.&#x0D; Gereç ve Yöntem: İki tane puberte öncesi dönemdeki (4 haftalık) dişi sıçanların ovaryumları küçük parçalara ayrılarak eksplant hücre kültürü oluşturuldu. Miks hücre kültüründe overyan stromal hücrelerle birlikte çoğalan ovaryum yüzey epiteli izole edilerek çoğaltıldı. Bununla birlikte ovaryum dokusunun histolojik kesitlerinde ovaryum yüzey epiteli incelenerek mikroskop altında görüntüleri alındı.&#x0D; Bulgular: Ovaryum yüzey epitelinin morfolojik görünümünün parke taşı (cobblestone) şeklinde olduğu görüldü. Faz kontrast mikroskobisi altında yapılan sayımda kültür kaplarında sırasıyla 2x106 ve 3x106 hücrenin ürediği izlendi. Petri kaplarının bazı alanlarında primordial folikül benzeri yapıların oluştuğu görüldü. Histolojik kesitlerde ise bazal membranın üzerinde primordial ve primer folikül öncüsü yapıların olduğu gözlemlendi.&#x0D; Sonuç: Hem hücre kültürlerinde, hem de histolojik kesitlerde oosit belirteçlerini (Gdf-9, C-Mos, Zpc, Stella) ve germ hücre belirteçlerini (Dazl,Vasa,Blimp1,Fragilis) göstermek bu hücrelerin farklılaşma kapasitelerini izlememiz açısından bizlere değerli bilgiler verebilir.