The Development of a Technique for Sensitizing Cells of Various Origins for Biotechnological Purposes

The functional activity stimulation of cell cultures was tested in MDBK cell culture, photobacteria AliivibriofischeriandHalobacteriumhalobium. Theaim of the investigation was to increase the ”yield” of the cells using an environmentallysafe stimulant and membrane-tropic agent that isalso safe for the experimenter. Ultrasonicwaves were used.Experimental ultrasonic exposure varied within the following limits: time from 1 to 300 sec, SATA-intensity of 0.01–2.0 W/cm2, generation frequency of 0.88 or 2.64 MHz, standing or traveling wave. The modulation frequency range was within 0.1–150 Hz. The devices used were: UST-1-01F, UST-5 and UST1.02C. The modulating generators were G3–112 and CP–110.Stimulation of MDBK cell growth was initiated by US-intensity of 0.03–0.05 W/cm2 , with an exposure of 5–30 sec.Exposure to ultrasound with an intensity of 0.2–0.4 W/cm2 (for 3 min) had a stimulating effect on bioluminescence and was associated with an increase in the growth rate ofA. fischeri. The findings indicated that 0.4 W/cm2ultrasonic intensity and modulation frequencies from 0.25 to 0.7 Hz can stimulate the growth of archaea.It was revealed that the maximum proliferation index in all cases of stimulant application was noted in cultures with minimal initial proliferative activity in the control.The authors expect thatthese results on the possibilities of acoustic continuous and modulated waves can be applied for biotechnological purposes to develop a new biotechnological method. Keywords: cell culture, ultrasound, proliferation, stimulation

Evaluating Large Spontaneous Deletions in a Bovine Cell Line Selected for Bovine Viral Diarrhea Virus Resistance

Bovine viral diarrhea virus’s (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genes PTPN12, GRID2, and RABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss of PTPN12, GRID2, or RABGAP1L gene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

Establishment of a Suspension MDBK Cell Line in Serum-Free Medium for Production of Bovine Alphaherpesvirus-1

The Madin–Darby bovine kidney (MDBK) cell line is currently used for the production of bovine alphaherpesvirus-1 (BoHV-1) vaccine. For the purpose of vaccine manufacturing, suspension cells are preferred over adherent ones due to simplified sub-cultivation and an easier scale-up process, both of which could significantly reduce production cost. This study aimed to establish a procedure for the culture of BoHV-1 in the suspended MDBK cell line in serum-free medium. We screened several commercially available serum-free media and chose ST503 for subsequent experiments. We successfully adapted the adherent MDBK cells to suspended growth in ST503 in the absence of serum. The maximum density of suspension-adapted MDBK cells could reach 2.5 × 107 cells/mL in ST503 medium with optimal conditions. The average size of suspension-adapted cells increased to 18 ± 1 µm from 16 ± 1 µm. Moreover, we examined tumorigenicity of the suspended cells and found no sign of tumorigenicity post adaptation. Next, we developed a protocol for the culture of BoHV-1 in the cell line described above and found that ultrasonic treatment could facilitate virus release and enhance virus yield by 11-fold, with the virus titer reaching 8.0 ± 0.2 log10TCID50/mL. Most importantly, the prototype inactivated BoHV-1 vaccine we generated using the suspension cultures of MDBK cells induced neutralizing antibodies to a titer comparable to that of the commercial inactivated BoHV-1 vaccine. Overall, we established and optimized a protocol for the production of inactivated BoHV-1 vaccine in MDBK cells adapted for suspension culture, which provides insights for future large-scale manufacturing of BoHV-1 vaccine.

Evaluating large spontaneous deletions in a bovine cell line selected for bovine viral diarrhea virus resistance - MDBK reads mapping to regions in genome not shared with CRIB cell line v1

Bovine viral diarrhea virus (BVDV) entry into bovine cells involves attachment of virions to cellular receptors, internalization, and pH-dependent fusion with endosomal membranes. The primary host receptor for BVDV is CD46; however, the complete set of host factors required for virus entry is unknown. The Madin-Darby bovine kidney (MDBK) cell line is susceptible to BVDV infection, while a derivative cell line (CRIB) is resistant at the level of virus entry. We performed complete genome sequencing of each to identify genomic variation underlying the resistant phenotype with the aim of identifying host factors essential for BVDV entry. Three large compound deletions in the BVDV-resistant CRIB cell line were identified and predicted to disrupt the function or expression of the genesPTPN12,GRID2, andRABGAP1L. However, CRISPR/Cas9 mediated knockout of these genes, individually or in combination, in the parental MDBK cell line did not impact virus entry or replication. Therefore, resistance to BVDV in the CRIB cell line is not due to the apparent spontaneous loss ofPTPN12,GRID2, orRABGAP1Lgene function. Identifying the functional cause of BVDV resistance in the CRIB cell line may require more detailed comparisons of the genomes and epigenomes.

IN VITRO ASSESSMENT OF THE BIOLOGICAL ACTIVITY OF A NEW REGENERATIVE AGENT PREPARED FROM THE CONCENTRATE OF DEPROTEINIZED DERMAL LAYER OF PORCINE SKIN

Background. Presently, a prospective direction for the development of regenerative medicine in the world is the search for regulatory molecules and the identification of molecular targets to stimulate the body's endogenous regenerative potential. The concentrate of the deproteinized dermal layer of porcine skin (СDDLPS) is a new therapeutic agent with restorative properties, the action of which is directed on the induction of the self resources of cells. Aim. The assessment of the effect of СDDLPS on the proliferative activity of mammalian cells of different histogenesis in vitro. Materials and Methods. To determine the amino acid composition of the СDDLPS liquid chromatography and biochemical methods were used. The biological effects and mechanisms of action of the drug were investigated by cell culture and molecular biological methods. The research was carried out using stable cell lines: human keratinocytes (HaCaT cell line), porcine endothelial cells (PAE cell line), bovine kidney cells (MDBK cell line) and mouse fibroblasts (3T3A31 cell line). Results. The cells of the bovine kidney MDBK cell line were the most sensitive to the effect of the CDDLPS. Also, the obtained results suggest that, depending on the concentration, the drug not only stimulates cell proliferation by 10–30 %, but also significantly enhances biosynthetic processes in cells, in particular, protein synthesis by 20–40 %. Conclusions. CDDLPS is an effective and affordable therapeutic agent with restorative properties, the biological activity of which manifests itself in the activation of cell biosynthetic and proliferative potentials and is comparable to effects of some growth factors, in particular epidermal growth factor

Correction of genetic instability of the genome by fractional irradiation of MDBK cells

With respect to given contradictory data in radiobiology, that according to some authors, irradiation of cells of plant and animal origin initiates DNA changes and according to others, repeated irradiation of cells in small doses has a stimulating effect on metabolism without increasing the purity of mutations, we conducted the present study, with the aim to study the stability of the genome of MDBK cell line against a background of single and double (fractional) irradiation. The object of the study was a continuous MDBK cell line (Madin-Darby Bovire Kidney Cell, cattle, kidney), which was grown in medium 199, manufactured by Federal State Budget Institution FCTRB-ARRVI (Kazan) with 10% bovine serum and the addition of antibiotics (benzylpenicillin sodium salt, kanamycin at 100 units/ml) (streptomycin sulfate at 100 μg/ml) according to standard procedure. A monolayer culture of MDBK cells in the logarithmic growth phase (3-4 days after culture) served as contacting cells. As a result, a radio-modified cell line, called MDBK-2, was obtained. Moreover, in an unirradiated MDBK cell culture, the level of chromosomal breakdowns was in the range of 1.03-3.1% with respect to the total number of studied anaphases. Based on the obtained data, it can be concluded, that during prolonged cultivation of MDBK cells, irradiated in a small (adaptive) dose and re-irradiated in 3-minute higher dose (5.95 Gy), there is a significant decrease in the yield of aberrant cells, induced by a test dose of γ-rays.

In-vivo Analgesic and In-vitro Cytoprotective Potential of Various Leaf Extracts of Bitter Apple

The present study investigated the potential of different leaf extracts of Bitter Apple (Citrullus colocynthis) as an analgesic agent and In-vitro cytoprotective ameliorative effects of the various extracts in thiomethoxam-induced toxicity in MDBK cell lines. Different leaf extracts of Citrullus colocynthis i.e. alcoholic, acetone and chloroform were investigated for analgesic activity at the dose rate of 50 mg/kg and 100 mg/kg in Wistar rats. For the assessment of analgesic activity, tail flick method was used. In-vitro cytoprotective activity of various leaf extracts (at concentrations of 5% and 10%) was evaluated in ATCC acquired MDBK cell lines and for this study, cytotoxicity was induced by thiomethoxam. For cytoprotective study, oxidative stress parameters- catalase, LPO, SOD and GPx were determined. Study on analgesic activity revealed the presence of dose dependent effect in all extracts with highest effect in alcoholic extract of Bitter Apple. It is believe that triterpene alkaloids and steroidal principles present in the plant products might be responsible for the analgesic effect.

Parameters for MDBK cell growth on microcarriers and BoHV-1 virus production

Bovine herpes virus 1 (BoHV-1) is an important veterinary agent , which causes infectious bovine rhinotra-cheitis. This disease affects the respiratory tract or genitals, causing weight loss, reduced milk production and abor-tion. Several vaccines against BoHV-1 have been developed. In this paper, we study the parameters for MDBK growth on microcarriers (Cytodex 1) and for BoHV-1 virus production. The cell culture attached to microcarriers is an effi-cient method to enlarge the surface of cell growth and for large-scale cell production. Our studies reveal that MDBK adhered to MCs in 30 minutes and that initial agitation of culture did not influence on the efficiency of adhesion or cell growth. In our experiments, we detected no relevant influence of agitation on initial cell adhesion of MDBK to MCs. The maximum cell yield was similar to all initial conditions of agitation studied. The maximum yield obtained in culture started with 15, 20 and 30 cells / MC, was respectively.8.7 x105, 9.3 x105 and 9.8 x105 cells / ml . The cellular distribution on the MCs at the beginning of the culture was more heterogeneous in higher initial densities. After three medium exchanges during MDBK cell culture, the increase in the final yield was 100% higher than that from culture performed without medium change (0.93 x 106 cells / mL). Replacing 50% of the culture medium with fresh medium after 24 hours of growth, the concentration of glucose (5 mM) and glutamine (1.8 mM) were almost completely res-tored. In these studies, BoHV-1 infections of MDBK were performed after 48, 72 and 86 hours with daily exchanges of 50% of the medium. The increase in viral titer was proportional to the number of viable cells present at the time of infection. The best result of BoHV-1 production was achieved when the infection was performed from 86 hours of cell culture, reaching about 3.7 x108 (TCID50/ml) after 24-48 hours of infection, being on average four times higher when compared to the culture in which the infection was performed with 48 hours of culture and approximately 2 times greater than the crop whose infection occurred after 72 hours of culture. The best yields are obtained when viral infections were performed on cultures with higher cell densities. The best result for the production of BoHV-1 occur-red with 10 MOI, 48 hours after infection, which yield was 24 and 41% superior to the MOI 0.1 and 1, respectively.