Invitro Cultivation Research Articles

Evaluating Bacterial Viability in Faecal Microbiota Transplantation: A Comparative Analysis of InVitro Cultivation and Membrane Integrity Methods.

Faecal microbiota transplantation (FMT) is a developing therapy for disorders related to gut dysbiosis. Despite its growing application, standardised protocols for FMT filtrate preparation and quality assessment remain undeveloped. The viability of bacteria in the filtrate is crucial for FMT's efficacy and for validating protocol execution. We compared two methods-in vitro cultivation and membrane integrity assessment-for their accuracy, reproducibility and clinical applicability in measuring bacterial viability in frozen FMT stool filtrate. Bacterial viability in stool filtrate was evaluated using (i) membrane integrity through fluorescent DNA staining with SYTO9 and propidium iodide, followed by flow cytometry and (ii) culturable bacteria counts (colony-forming units, CFU) under aerobic or anaerobic conditions. Using different types of samples (pure bacterial culture, stool of germ-free and conventionally bred mice, native and heat-treated human stool), we refined the bacterial DNA staining protocol integrated with flow cytometry for assessment of bacterial viability in frozen human stool samples. Both the membrane integrity-based and cultivation-based methods exhibited significant variability in bacterial viability across different FMT filtrates, without correlation. The cultivation-based method showed a mean coefficient of variance of 30.3%, ranging from 7.4% to 60.1%. Conversely, the membrane integrity approach yielded more reproducible results, with a mean coefficient of variance for viable cells of 6.4% ranging from 0.2% to 18.2%. Bacterial viability assessment in stool filtrate using the membrane integrity method offers robust and precise data, making it a suitable option for faecal material evaluation in FMT. In contrast, the cultivation-dependent methods produce inconsistent outcomes.

A SIMPLE, QUICK, AND ECONOMICAL METHOD FOR IN VITRO CULTIVATION OF ECHINOCOCCUS MULTILOCULARIS METACESTODE AND GENERATION OF PRIMARY CELLS.

Alveolar echinococcosis is considered to be one of the most potentially lethal parasitic zoonotic diseases. However, the molecular mechanisms by which Echinococcus multilocularis interacts with hosts are poorly understood, hindering the prevention and treatment of this disease. Due to the great advantages of cell culture systems for molecular research, numerous attempts have been made to establish primary cell cultures for E. multilocularis. In this study we developed a simple, rapid, and economical method that allows E. multilocularis metacestode tissue blocks to generate daughter vesicles without the continuous presence of host feeder cells in a regular medium. We performed anaerobic, hypoxic (1% O2), normoxic, and semi-anaerobic (in sealed tubes) cultures and found that E. multilocularis metacestode tissues can produce daughter vesicles only in the sealed tubes after 4 wk of incubation. The daughter vesicles cultivated in this system were remarkably enlarged under anaerobic conditions after 8 days of culture, whereas vesicles cultured under hypoxic (1% O2) and normoxic conditions showed only a mild increase in volume. Our in vitro cultivated vesicles showed strong viability and could be used to test antiparasitic drugs, isolate primary cells, and infect animals.

Spinner Flask Bioreactor in Tissue Engineering

Spinner Flask Bioreactors are usually made up of glass or plastic vessel which have been widely used in Tissue Engineering from production of articular cartilage to production of osteoblast cells that help in bone regeneration. Cartilage grown in spinner flask bioreactors had more cells and less GAG than the other types of bioreactors in tissue engineering. In recent years, these bioreactors have also been used for the invitro cultivation of human tenocytes and MSCs. In this type of bioreactor, the cell/scaffold constructs are connected to vertical needles striking from pinnacle of the vessel and immersed inside the culture medium. The pinnacle or the top part of this bioreactor is used for gas exchange and medium oxygenation. Mixing of the medium is maintained with a stir bar at the lowest of the vessel or different blending mechanisms. Spinner Flask Bioreactors have grabbed increased attention in recent years due to its wide range of Tissue Engineering applications. In this review, we have tried to explore the different domains where these bioreactors have found enhanced applications. Keywords: Spin Flask Bioreactor Tissue Engineering, Cell Seeding, Bone Tissue Engineering, Articular Cartilage

May the dark be with roots: a perspective on how root illumination may bias invitro research on plant-environment interactions.

Roots anchor plants to the soil, providing them with nutrients and water while creating a defence network and facilitating beneficial interactions with a multitude of living organisms and climatological conditions. To facilitate morphological and molecular studies, root research has been conducted using invitro systems. However, under natural conditions, roots grow in the dark, mainly in the absence of illumination, except for the relatively low illumination of the upper soil surface, and this has been largely ignored. Here, we discuss the results found over the last decade on how experimental exposure of roots to light may bias root development and responses through the alteration of hormonal signalling, cytoskeleton organization, reactive oxygen species or the accumulation of flavonoids, among other factors. Illumination alters the uptake of nutrients or water, and also affects the response of the roots to abiotic stresses and root interactions with the microbiota. Furthermore, we review invitro systems created to maintain roots in darkness, and provide a comparative analysis of root transcriptomes obtained with these devices. Finally, we identify other experimental variables that should be considered to better mimic soil conditions, whose improvement would benefit studies using invitro cultivation or enclosed ecosystems.

In vitro culture of Mesocestoides corti metacestodes and isolation of immunomodulatory excretory-secretory products.

Cestode-mediated diseases hold the interesting feature of persisting metacestode larvae dwelling within the host tissues, in the midst of the immune response. Excretory-secretory (ES) products of the metacestode larval stage modulate the host immune response and modify the outcome of the disease. Therefore, isolation and analysis of axenic metacestode ES products are crucial to study their properties. Here, we report the development of a system for long-term invitro cultivation of the metacestode of the parasitic cestode Mesocestoides corti (syn. Mesocestoides vogae). Although feeder cells and host serum supported the early growth of the parasite, long-term survival was not dependent on host serum or host-derived factors enabling the collection of parasite released products in serum-free medium. Functionally, these axenic ES products recapitulated M.corti tetrathyridia's ability to inhibit LPS-driven IL-12p70 secretion by dendritic cells. Thus, our new axenic culture system will simplify the identification and characterization of M.corti-derived immunomodulatory factors that will indirectly enable the identification and characterization of corresponding factors in the metacestode larvae of medically relevant cestodes such as Echinococcus multilocularis that are not yet amenable to serum-free cultivation.

In Vitro Expression of the Extracellular Matrix Components Aggrecan, Collagen Types I and II by Articular Cartilage-Derived Chondrocytes.

The extracellular matrix (ECM) of hyaline cartilage is perfectly suited to transmit articular pressure load to the subchondral bone. Pressure is transferred by a high amount of aggrecan-based proteoglycans and collagen type II fibres in particular. After any injury, the hyaline cartilage is replaced by fibrocartilage, which is low in proteoglycans and contains collagen type I predominantly. Until now, long-term results of therapeutic procedures including cell-based therapies like autologous chondrocyte transplantation (ACT) lead to a replacement tissue meeting the composition of fibrocartilage. Therefore, it is of particular interest to discover how and to what extent isolation and invitro cultivation of chondrocytes affect the cells and their expression of ECM components. Hyaline cartilage-derived chondrocytes were cultivated invitro and observed microscopically over a time period of 35days. The expression of collagen type I, collagen type II and aggrecan was analysed using RT-qPCR and Western blot at several days of cultivation. Chondrocytes presented a longitudinal shape for the entire cultivation period. While expression of collagen type I prevailed within the first days, only prolonged cultivation led to an increase in collagen type II and aggrecan expression. The results indicate that chondrocyte isolation and invitro cultivation lead to a dedifferentiation at least to the stage of chondroprogenitor cells.

Real-time proliferation of porcine cumulus cells is related to the protein levels and cellular distribution of Cdk4 and Cx43

The proper maturation of cumulus somatic cells depends on bidirectional communication between the oocyte and the surrounding cumulus cells (CCs). The aim of this study was (i) to investigate maturation markers, such as Cx43 and Cdk4 protein levels, and (ii) to analyze the distribution of these two proteins in CCs cultured for 44, 88, 132, and 164 hours in both separated and cumulus-enclosed oocyte cultures. CCs were isolated from porcine ovarian follicles after the treatment of the recovered COCs with collagenase. Then, the separated CCs were cultured in TCM-199 for 0 to 164 hours, using a real-time cellular analyzer; however, the immunostaining was performed only after 44, 88, and 132 hours. The protein levels and distribution were analyzed using confocal microscopy. After the CCs underwent in vitro cultivation (IVC) for 25 hours, a logarithmically increasing normalized proliferation index was found throughout the entire 164 hours cultivation time. The Cx43 and Cdk4 proteins were observed at higher levels after 44 hours of culture than before IVC. After 88 and 132 hours of IVC, no significant alterations in either mRNA or protein levels of Cx43 and Cdk4 were found. Cx43 and Cdk4 were localized in the cell nucleus before IVC, whereas after 44, 88, and 132 hours of IVC, both proteins translocated to the cytoplasm. In cumulus-enclosed oocyte cultures, Cdk4 was localized both in the nucleus and cytoplasm, whereas Cx43 was only in the cytoplasm. Additionally, only low levels of the cumulus expansion markers MIS and SNAT3 were observed. In summary, we could demonstrate that the in vitro cultivation of CCs was associated with cell proliferation and that Cx43 and Cdk4 gene expression was upregulated after IVC, resulting in significantly higher protein levels. Moreover, the two proteins translocated from the nucleus to the cytoplasm of the CCs during IVC. The protein distribution is presumably related to different protein functions during bidirectional communication via gap junction communication.

Short-term Cultivation of Porcine Cumulus Cells Influences the Cyclin-dependent Kinase 4 (Cdk4) and Connexin 43 (Cx43) Protein Expression—A Real-time Cell Proliferation Approach

The CC (cumulus cell) proliferation index in relation to the expression and distribution of Cdk4 and Cx43 proteins, which are crucial factors for oocyte maturation, was investigated. Cumulus-oocyte complexes (COCs) were recovered from pubertal crossbred Landrace gilts and treated with collagenase, and separated CCs were cultured in standard TCM199 medium for 44 h. At each step of in vitro cultivation (IVC) of CCs (0, 12, 24 and 44 h), a normalized proliferation index was assessed. Cdk4 and Cx43 protein expression and the CC-specific cellular distribution were analyzed by confocal microscopic observation. The normalized proliferation index (number of cells attached, measured by impedance) was increased in the first 12 h of IVC (P<0.01) and differed between 12 h and 24 h of cultivation (P<0.001). Later, between 24 h–44 h of IVC, the CC proliferation rate was stable, and no significant differences were observed. Based on the confocal microscopic observation, increased expression of both Cdk4 and Cx43 was found after 44 h of IVC compared with the expression of these proteins before IVC. Moreover, after IVC, a substantial translocation of Cdk4 and Cx43 was noted from the nucleus to the cytoplasm of CCs. In conclusion, it was demonstrated for the first time that CCs can be cultured in vitro separately without oocytes and that the proliferation index was significantly increased in the first 12 h of IVC, which may reflect the process of ordinary cumulus cell expansion. Furthermore, the expression of both Cdk4 and Cx43 in CCs suggested that these proteins may be regarded as markers not only of proper oocyte maturation but also of CC differentiation. Translocation of these proteins into the cytoplasm of CCs after 44 h of IVC may be related to the expansion process.

Studies on production of excretory-secretoryproducts from 3 rdstage larvae of gnathostoma spinigerum by an invitro cultivation

Studies on production of excretory-secretoryproducts from 3 rdstage larvae of gnathostoma spinigerum by an invitro cultivation

Inhibition of malaria parasite invasion into erythrocytes pretreated with membrane-active drugs.

Normal rhesus monkey erythrocytes were incubated with various membrane-active drugs (for 1 h at 37 degrees C) and after thorough washing, were exposed to infection with Plasmodium knowlesi in an invitro cultivation system. The ability of merozoites to infect with drug-pretreated erythrocytes was assessed both by counting the number of infected cells and by measuring the incorporation of [3H]isoleucine into parasite protein. Marked inhibition of invasion was observed with vinblastine and colchicine, at concentrations greater than 5 x 10(-4) M, respectively. At similar concentrations, cytochalasin B or amantadine had no apparent effect. The addition of 10(-3) M 3',5'-cAMP to the medium during the incubation in the presence of colchicine or vinblastine partially decreased the inhibitory effects. The effects of colchicine and vinblastine on parasite invasion may be correlated with a reversible alteration in erythrocyte conformation (spherocytosis) which occurs at similar drug concentrations to those above and which can be relieved by simultaneous incubation with cAMP. A possible mechanism of action is proposed.

Nuclear and cell divisions in isolated pollen of Petunia hybrida in agar suspension cultures.

IN VITRO cultivation of anthers leading to androgenesis provides a new approach to the production of angiosperms possessing the gametophytic number of chromosomes. Kameya and Hinata1 reported cell cluster formation from isolated pollen grains of Brassica oleracea. This study explores the use of isolated Petunia pollen to study the conditions leading to vegetative growth.