Proliferation and Differentiation of Intestinal Caco-2 Cells Are Maintained in Culture with Human Platelet Lysate Instead of Fetal Calf Serum

Cell lines are widely used as in vitro model systems and substitute for animal experiments. The frequently used Caco-2 cell line is considered to reflect characteristics of differentiated intestinal epithelium. However, the need to culture the cells with fetal calf serum (FCS) induces a high variability, risk of contamination and is ethically disputed. We tested the culture of Caco-2 cells with human platelet lysate (PL) instead of FCS. We compared cell viability and differentiation by measuring ATP levels, gene and protein expression of specific markers in total cell extracts, brush border membrane vesicles (BBM) and lipid rafts (LR). Cell viability was slightly enhanced in cells grown with PL compared to FCS. The cells differentiated to an intestinal phenotype like the cells cultured in FCS, as indicated by the similar gene expression levels of hexose and protein transport proteins and the structural protein VILLIN. BBM showed a comparable distribution of the intestinal hydrolases, indicating a maintained cell membrane polarity. The distribution of the marker protein FLOTILLIN-2 in LR was also similar. We conclude that PL is an exquisite and suitable replacement for FCS in the culture of Caco-2 cells that can eliminate many disadvantages incurred due to the use of FCS.

Direct addition of poly-lysine or poly-ethylenimine to the medium, a simple alternative to plate pre-coating

For most cell culture experiments, it is indispensable that the cells are firmly anchored to the culture plates, tolerating several rinsing steps, and withstanding shear forces or temperature changes without detaching. For semi-adherent cells such as the very common HEK 293 cells, this could so far be obtained only by time-consuming plate pre-coating with cationic polymer solutions. We report here, that i) pre-coating with the cheaper poly-ethylenimine (PEI) works as well as the commonly used poly-D-lysine (PDL), but more importantly and novel ii) that simple direct addition of either PEI (1.5 µg/ml) or PDL (2 µg/ml) to the cell culture medium results in strongly anchored HEK 293 cells, indistinguishable from ones seeded on pre-coated plates. Therefore, the replacement of plate pre-coating by direct addition of either PEI or PDL gives comparable excellent results, but is highly labour-, time-, and cost-efficient. Interestingly, additional experiments in this context showed that strong cell attachment requires only cationic polymers but not fetal calf serum added to the medium. Fetal calf serum is, however, of course required for further maintenance and growth of the cells.

The effects of Glucose and Ascorbic acid on in vitro development of Echinococcus granulosus Metacestodes

Echinococcus granulosus-developed metacestodes in the cultured medium are used for the assessment of its susceptibility to different compounds; however, this procedure is time-consuming and risky. In the present study, aspirated protoscoleces from the infected sheep were used to evaluate the effects of glucose, as an energy source, as well as ascorbic acid, as an antioxidant vitamin, on larval development. Protoscoleces were maintained in RPMI1640 culture media containing 10% fetal calf serum, as well as different concentrations of glucose (6 and 8 mg/ml) and ascorbic acid (25, 50, and 100 µg/ml). A culture medium containing 4 mg/ml of glucose was served as the control. Larger cysts were achieved in a shorter time from the medium enriched with 6 mg/ml of glucose (740 ± 20 µm) compared to the control group (420 ± 40 µm). However, in the groups treated with ascorbic acid, the number of cysts was higher in 100 µg/ml (32.5 ± 0.7) compared to the control group (12.5 ± 0.7). Additionally, the mature cysts were achieved on the 7th day of cultivation with 100 µg/ml of ascorbic acid compared to 18 days in the control group.

Inactivation of Candida auris and Candida albicans by ultraviolet-C

We evaluated the ability of an ultraviolet-C (UV-C) room decontamination device to kill Candida auris and C. albicans. With an organic challenge (fetal calf serum), the UV-C device demonstrated the following log10 reductions for C. auris of 4.57 and for C. albicans of 5.26 with direct line of sight, and log10 reductions for C. auris of 2.41 and for C. ablicans of 3.96 with indirect line of sight.

MO583IN VITRO AND EX VIVO EXPERIMENTS OF VASCULAR CALCIFICATION

Background and Aims Vascular calcification (VC) is one major complication in patients with chronic kidney disease whereas a misbalance in calcium and phosphate metabolism plays a crucial role. The mechanisms underlying VC have not been entirely revealed to date. Therefore are the studies aiming at the identification and characterization of the mediators/uremic toxins involved in VC ongoing and highly relevant. However, currently many different protocols being used in the studies of vascular calcification processes. This complicates the comparison of study outcomes, composing systematic reviews, and meta-analyses. Moreover, the reproducibility of data is hampered, and the efficiency in calcification research through the lack of a standardized protocol is reduced. In this study, we developed a standardized operating protocol for in vitro and ex vivo approaches to aiming at the comparability of these studies. Method We analysed in vitro and ex vivo experimental conditions to study VC. Vascular smooth muscle cells (HAoSMCs) were used for in vitro experiments and aortas from Wistar rats were used for ex vivo experiments. The influence of the following conditions was studied in detail: • Phosphate and calcium concentrations in calcifying media. • Incubation time. • Fetal calf serum (FCS) concentration. The degree of calcification was estimated by quantification of calcium concentrations that were normalized to protein content (in vitro) or to the dry weight of the aortic ring (ex vivo). Additionally, the aortic rings were stained using the von Kossa method. Optimal conditions for investigating medial vascular calcification were detected and summarized in the step-by-step protocol. Results We were able to demonstrate that the degree and the location of VC in vascular smooth muscle cells and aortic rings were highly dependent on the phosphate and CaCl2 concentration in the medium as well as the incubation time. Furthermore, the VC was reduced upon increasing fetal calf serum concentration in the medium. An optimized protocol for studying vascular calcification in vitro and ex vivo was developed and validated. The final protocol (Figure 1) presented will help to standardize in vitro and ex vivo approaches to investigate the processes of vascular calcification. Conclusion In the current study, we developed and validated a standardized operating protocol for systematic in vitro and ex vivo analyses of medial calcification, which is essential for the comparability of the results of future studies.

Synovial Fluid Profile Dictates Nanopracticle Uptake into Cartilage- Implications of the Protein Corona for Novel Arthitis Treatments

<div>Intra-articular drug delivery strategies aiming to deliver drugs in diseases affected by cartilage-related issues are using electrostatic interactions to penetrate the dense cartilage matrix. This enables delivery of sufficient drug concentrations to the chondrocytes to mediate the desired therapeutic effect. As it is well known that size and charge of nanoparticles affects its interactions with the surrounding biological fluids, where proteins adsorb to the NP surface, resulting in a protein corona. There are, however, no studies investigating how the formed protein coronas affect cartilage uptake and subsequent cellular uptake, nor how they affect other cells present in the synovium of such diseases. Here, we explore the differences between the protein coronas that form when NP are incubated in synovial fluid from osteoarthritic and rheumatoid arthritis patients and compare this to results obtained using fetal calf serum (FCS), as guide for researchers working on joint drug delivery. We demonstrate that the protein corona indeed affects the uptake into cartilage, where there are major differences between the model proteins in fetal calf serum, as compared to synovial fluid from rheumatoid arthritis patients as well as osteoarthritis patients. The data suggests that when developing drug delivery vehicles for joint diseases that leverages electrostatic interactions and size, the interactions with proteins in the biological milieu is highly relevant to consider.</div>

Synovial Fluid Profile Dictates Nanopracticle Uptake into Cartilage- Implications of the Protein Corona for Novel Arthitis Treatments

<div>Intra-articular drug delivery strategies aiming to deliver drugs in diseases affected by cartilage-related issues are using electrostatic interactions to penetrate the dense cartilage matrix. This enables delivery of sufficient drug concentrations to the chondrocytes to mediate the desired therapeutic effect. As it is well known that size and charge of nanoparticles affects its interactions with the surrounding biological fluids, where proteins adsorb to the NP surface, resulting in a protein corona. There are, however, no studies investigating how the formed protein coronas affect cartilage uptake and subsequent cellular uptake, nor how they affect other cells present in the synovium of such diseases. Here, we explore the differences between the protein coronas that form when NP are incubated in synovial fluid from osteoarthritic and rheumatoid arthritis patients and compare this to results obtained using fetal calf serum (FCS), as guide for researchers working on joint drug delivery. We demonstrate that the protein corona indeed affects the uptake into cartilage, where there are major differences between the model proteins in fetal calf serum, as compared to synovial fluid from rheumatoid arthritis patients as well as osteoarthritis patients. The data suggests that when developing drug delivery vehicles for joint diseases that leverages electrostatic interactions and size, the interactions with proteins in the biological milieu is highly relevant to consider.</div>