Triple Culture Research Articles

Development of bacterial bioformulations using response surface methodology.

Bacterial consortia exhibiting plant growth promoting properties have emerged as a sustainable approach for crop improvement. As the main challenge associated with them is viability loss and performance under natural conditions, a robust approach for designing bioformulation is needed. In this study, an efficient bioformulation was developed using spontaneous mutants of three bacterial strains for growth promotion of Cajanus cajan. Optimization of additives for solid [carboxymethylcellulose (CMC), and glycerol) and liquid [polysorbate, CMC, and polyvinyl pyrrolidone (PVP)] bioformulations was done by Response Surface Methodology using Central Composite Design. The stability of each bioinoculant in the formulation was assessed at 30°C and 4°C. The efficiency of the liquid bioformulation was checked in planta in sterile, and subsequently in non-sterile, soil. The maximum cell count was observed in solid bioformulation with 0.1g L-1 CMC and 50% glycerol (8.10×108, 3.69×108, and 7.39×108 for Priestia megaterium, Azotobacter chroococcum, and Pseudomonas sp. SK3, respectively) and in liquid bioformulation comprising 1% PVP, 0.1g L-1 CMC, and 0.025% polysorbate (8×109, 3.8×109, and 6.82×109 for Priestia megaterium, Azotobacter chroococcum, and Pseudomonas sp. SK3, respectively). The bioinoculants showed a higher viability (6 months) at 4°C compared to 30°C. Triple culture consortia enhanced plant growth in comparison to the control. The strains could be detected in soil till 45 days after sowing. The study established a systematic process for developing a potent bioformulation to promote agricultural sustainability. Using mutant strains, the bioinoculants could be tracked. In planta assays revealed that the triple culture consortium out-performed mono and dual cultures in terms of impact on plant growth.

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Developing a Triple Cell Culture Model of the Human Blood-Brain Barrier

Development and characterisation of a novel complex triple cell culture model of the human alveolar epithelial barrier

Owing to increased pressure from ethical groups and the public to avoid unnecessary animal testing, the need for new, responsive and biologically relevant in vitro models has surged. Models of the human alveolar epithelium are of particular interest since thorough investigations into air pollution and the effects of inhaled nanoparticles and e-cigarettes are needed. The lung is a crucial organ of interest due to potential exposures to endogenous material during occupational and ambient settings. Here, an in vitro model of the alveolar barrier has been created in preparation for use in the quasi-air liquid interface (qALI) and (aerosol) air–liquid interface (ALI) exposures. The model consists of an alveolar type 1-like cell line (TT1), an alveolar type 2-like cell line (NCI-H441) and a model of (alveolar) macrophages (dTHP-1). The model formulates a complex, multi-cellular system, cultured at the air–liquid interface, that mimics the apical layer of the alveolar epithelial region in the human lung. Characterisation data has shown that both TT1 and NCI-H441 epithelial cells are able to be cultured together in addition to dTHP-1 cells through imaging (morphology), pro-inflammatory response and viability measurements. This dataset also demonstrates evidence of a reasonable barrier created by the cell culture in comparison to negative controls. Furthermore, it shows that while maintaining a low baseline of (pro)-inflammatory mediator expression during normal conditions, the model is highly responsive to inflammatory stimuli. This model is proposed to be suitable for use in toxicology testing of inhaled exogenous agents.

Hyperglycaemia perturbs blood-brain barrier integrity through its effects on endothelial cell characteristics and function

ABSTRACT Breakdown of blood-brain barrier (BBB) represents a key pathology in hyperglycemia-mediated cerebrovascular damage after an ischemic stroke. As changes in the level and nature of vasoactive agents released by endothelial cells (ECs) may contribute to BBB dysfunction, this study first explored the specific impact of hyperglycemia on EC characteristics and secretome. It then assessed whether secretome obtained from ECs subjected to normoglycaemia or hyperglycemia might regulate pericytic cytokine profile differently. Using a triple cell culture model of human BBB, composed of brain microvascular EC (BMEC), astrocytes and pericytes, this study showed that exposure to hyperglycemia (25 mM D-glucose) for 72 h impaired the BBB integrity and function as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux of sodium fluorescein. Dissolution of zonula occludens-1, a tight junction protein, and appearance of stress fibers appeared to play a key role in this pathology. Despite elevations in angiogenin, endothelin-1, interleukin-8 and basic fibroblast growth factor levels and a decrease in placental growth factor levels in BMEC subjected to hyperglycemia vs normoglycaemia (5.5 mM D-glucose), tubulogenic capacity of BMECs remained similar in both settings. Similarly, pericytes subjected to secretome obtained from hyperglycemic BMEC released higher quantities of macrophage migration inhibitory factor and serpin and lower quantities of monocyte chemoattractant protein-1, intercellular adhesion molecule, interleukin-6 and interleukin-8. Taken together these findings indicate the complexity of the mechanisms leading to BBB disruption in hyperglycemic settings and emphasize the importance of endothelial cell-pericyte axis in the development of novel therapeutic strategies.

Cellular response of advanced triple cultures of human osteocytes, osteoblasts and osteoclasts to high sulfated hyaluronan (sHA3)

Bone remodelling, important for homeostasis and regeneration involves the controlled action of osteoblasts, osteocytes and osteoclasts. The present study established a three-dimensional human in vitro bone model as triple culture with simultaneously differentiating osteocytes and osteoclasts, in the presence of osteoblasts. Since high sulfated hyaluronan (sHA3) was reported as a biomaterial to enhance osteogenesis as well as to dampen osteoclastogenesis, the triple culture was exposed to sHA3 to investigate cellular responses compared to the respective bone cell monocultures. Osteoclast formation and marker expression was stimulated by sHA3 only in triple culture. Osteoprotegerin (OPG) gene expression and protein secretion, but not receptor activator of NF-κB ligand (RANKL) or sclerostin (SOST), were strongly enhanced, suggesting an important role of sHA3 itself in osteoclastogenesis with other targets than indirect modulation of the RANKL/OPG ratio. Furthermore, sHA3 upregulated osteocalcin (BGLAP) in osteocytes and osteoblasts in triple culture, while alkaline phosphatase (ALP) was downregulated.

The behavior of the Bacillus probiotic species under conditions of co-cultivation

Combining probiotic bacteria is a promising strategy to increase the effectiveness and avoid side effects of probiotic therapy. Bacteria that find themselves in a common environment are able to both change their properties and show new ones under the influence of each other. The change of colonization and antagonistic properties, which provide bacteria with competitive advantages in the development of new spaces, deserves special attention.
 The aim of this research was to study the features of the mutual influence of probiotic bacilli: B. clausii, B. coagulans and B. subtilis on growth, swimming and swarming motility when co-cultivated on agar media of different solidity.
 Materials and methods. The study used commercial strains of bacilli from three probiotic preparations: Enterogermina, Lactovit forte and Subalin. The ability of the studied species of bacilli to influence each other’s growth was investigated by the agar block method (using 1.5 % nutrient agar) and the spot-on-lawn assay (using 0.7 % nutrient agar). The study of the mutual influence on swimming and swarming activity was carried out when bacilli were cultivated on 0.25 % and 0.70 % tryptone agar, respectively. The diameters of swimming halos and migration swarms formed by monoand mixed cultures were measured, and the areas covered by them were compared. The phenotypes of the meeting of swarms formed by cultures spotted on the swarm plates at different locations were also investigated.
 Results. The studied probiotic species of the bacilli did not show a strong ability to inhibit each other’s growth. The B. clausii culture had no inhibitory effect, and the B. coagulans culture demonstrated a moderate inhibitory influence on the growth of the other two species of bacilli when using both diffusion methods. The B. subtilis culture showed moderate or weak inhibitory activity against the B. clausii culture and weak or no inhibitory activity against the B. coagulans culture using the agar block method or spot-on-lawn assay, respectively. The B. coagulans + B. subtilis, B. clausii + B. coagulans and B. clausii + B. subtilis mixed cultures showed moderate, weak and no inhibitory activity against third cultures, respectively. The studied species of bacilli showed different swimming ability and swarming potential as well as the ability to influence each other’s motility. Swimming halos formed by the B. clausii + B. subtilis and B. clausii + B. сoagulans + B. subtilis mixed cultures covered significantly larger plate areas than the swimming halos formed by each culture separately during the same cultivation time. The highest swarming potential was observed in B. coagulans culture and B. cla usii + B. coagulans + B. subtilis mixed culture. The studied bacilli did not show the ability to merge swarms, but, on the contrary, their swarms at the point of contact formed visible “boundary” or “intermediate” lines, demonstrating the ability to identify nonself.
 Conclusions. The obtained results indicate the ability of the probiotic species of bacilli: B. clausii, B. coagulans and B. subtilis to mutually influence growth, swimming and swarm motility. The highest indicators of swimming and swarming of the triple mixed culture indicate an increase in the colonization potential of the studied bacilli when they are combined. The hypothesis of mutual induction of inhibitory compounds production by bacilli, which may increase the overall antagonistic potential of the triple mixed culture, is subject to further confirmation.

Can daily bathing with 4% chlorhexidine + daily chlorhexidine wipe for 1week be effective in decolonizing Candida auris colonization?

Herein, it is aimed to present the decolonizing rates of Candida auris colonized cases after daily bathing with 4% chlorhexidine plus daily cleaning with 4% chlorhexidine wipe for 1week (will be mentioned as DCHX). The study period was from October, 2021, to November, 2022. Inclusion criteria were (i) age > 18, (ii) receiving DCHX, (iii) proven C. auris carrier on auricular, or axillar or inguinal swab surveillance cultures up to 5-day period before DCHX. Cases with three consecutive negative surveillance cultures 3days apart were considered to be decolonized. A total of 38 cases [14 female, aged 61.8 ± 15.5years] fulfilled the inclusion criteria. Six (15.8%), 23 (60.1%), and 22 cases (57.8%) were postauricular, inguinal, and axillary culture positive, respectively. Only three cases (7.9%) were triple culture positive. Nine cases (23.7%) had three consequent negative surveillance cultures after DCHX and were considered to be decolonized.There was no significant difference in decolonization rates of concomitant only antibiotic receiving cohort vs. concomitant antifungal + antibiotic receiving cohort (5/16 vs. 2/8, p = 1) were decolonized similarly. Of the nine C. auris decolonized cases, two developed C. auris infection in 30days follow-up after decolonization. However, 10 (34.5%) of 29 non-decolonized cases developed C. auris infection (p: 0.450) within 30days after surveillance culture positivity. Over all cohorts, day 30 mortality was 23.7% (9/38). In conclusion, based on our observational and relatively small uncontrolled series, it appears that DCHX is not very effective in decolonizing C. auris carriers (especially in cases who are C. auris colonized in > 1 areas), although it is not completely ineffective.

Are multiple blood cultures advantageous for canine patients?

Successful treatment of bacteremic patients depends largely on timely detection of blood-borne pathogens. Failure to detect an infection and/or contamination of blood samples can substantially delay the proper treatment. To increase the detection rate of blood-borne pathogens, well-established guidelines on blood collection and processing have been practiced in human medicine. Investigations involving human blood cultures have shown that the multiple blood sample approach significantly improves the detection rate of bacterial pathogens in the blood. Unfortunately, veterinary-specific blood culture guidelines have not been defined. Therefore, we compared detection rates of blood-borne pathogens between single and multiple blood culture approaches in a retrospective study of the clinical data from canine blood culture cases. We analyzed the data that had been collected over ~6 y and 8 mo from 177 dogs admitted to a veterinary medical teaching hospital. The triple blood culture approach increased the detection rate of blood-borne pathogens by 19.5% compared to single sampling. The optimal timing between multiple sample collections remains to be determined.

In vitro triple culture model of retinoblastoma for pre-clinical investigations.

Retinoblastoma (Rb) is a rare cancer of the retina that occurs during early childhood. The disease is relatively rare but aggressive, accounting for ∼3% of childhood cancers. Treatment modalities encompass the administration of large doses of chemotherapeutic drugs, which result in multiple side-effects. Therefore, it is essential to have safe and effective newer therapies and suitable physiologically relevant, alternative-to-animal, in vitro cell culture-based models to enable rapid and efficient evaluation of potential therapies. This investigation was focused on the development of a triple co-culture model comprising Rb, retinal epithelium, and choroid endothelial cells, using a protein coating cocktail, to recapitulate this ocular cancer under in vitro conditions. This resulting model was used for screening drug toxicity, based on the growth profile of Rb cells, using carboplatin as the model drug. Further, a combination of bevacizumab and carboplatin was evaluated using the developed model, to lower the concentration of carboplatin and thereby reduce its physiological side-effects. The effect of drug treatment on the triple co-culture was assessed by increase in the apoptotic profile of Rb cells. Further, the barrier properties were found to be lower with a decrease in the angiogenetic signals that included expression of vimentin. Measurement of cytokine levels signified reduced inflammatory signals due to the combinatorial drug treatment. These findings validated that the triple co-culture Rb model was suitable for evaluating anti-Rb therapeutics and could thereby decrease the immense load on animal trials, which are the primary screens employed for evaluating retinal therapies.

Pericytes as an Essential Part in Transwell Models of the BBB <i>in Vitro</i>

In this study we aimed to demonstrate the advantages of using a quadruple culture model of the blood-brain barrier (BBB) in vitro in comparison with a common triple culture model, as well as to show the impact of pericytes on endothelial cells of the BBB. We co-cultured primary rat brain microvascular endothelial cells (BMECs), pericytes, astrocytes and neurons in a Transwell BBB model in vitro. Then, we carried out quantitative analysis to compare transendothelial electrical resistance (TEER) values, as well as expression levels of tight junction proteins, ZO1 and JAM1, in the triple culture and the quadruple culture Transwell BBB models in vitro. According to the obtained data, the quadruple culture model of the BBB in vitro has advantages over the triple culture model, since the presence of pericytes is accompanied by higher TEER values and higher expression levels of tight junction proteins in endothelial cells. The results presented in the study are consistent with the world scientific literature and confirm the hypothesis that pericytes not only offer mechanical support for endothelial cells, but also play a key role in signaling networks between different cell types of the neurovascular unit (NVU) and thus regulate the barrier functions of the BBB. According to this, co-culture of BMECs, astrocytes, and neurons with pericytes is essential for BMECs optimum phenotype and offers a closer representation of the in vivo environment.

An alternative in vitro model considering cell-cell interactions in fiber-induced pulmonary fibrosis

Particularly since the wide-ranging health effects of asbestos exposure became known, great emphasis has been placed on detailed toxicity testing of known but also newly developed fiber materials. Exposure to respirable pollutants like fibers can lead to tissue injury causing lung diseases such as pulmonary fibrosis or cancer. In order to detect the toxic potential of such aerosols at an early stage, the development of suitable test systems is essential. In this study, we illustrate the development of an advanced in vitro cell model closely resembling the physiological structure of the alveoli, and we highlight its advantages over simpler models to predict pro-fibrotic changes. For this reason, we analyzed the cytotoxic effects of fiber-like multi-walled carbon nanotubes after 24 and 48 h exposure, and we investigated inflammatory, genotoxic and pro-fibrotic changes occurring in the developed triple culture consisting of lung epithelial cells, macrophages and fibroblasts compared to a co-culture of epithelial cells and fibroblasts or a mono culture of epithelial cells. In summary, the triple culture system is more precisely able to detect a pro-fibrotic phenotype including epithelial-mesenchymal transition as well as secondary genotoxicity, even if exhibiting lower cytotoxicity in contrast to the less advanced systems. These effects might be traced back to the complex interplay between the different cell types, all of which play an important role in the inflammatory response, which precedes wound healing, or even fibrosis or cancer development.

A Triple Primary Cell Culture Model of the Human Blood-Brain Barrier for Studying Ischemic Stroke In Vitro.

Ischemic stroke is a major cause of death and disability worldwide with limited therapeutic options. The neuropathology of ischemic stroke is characterized by an interruption in blood supply to the brain leading to cell death and cognitive dysfunction. During and after ischemic stroke, blood-brain barrier (BBB) dysfunction facilitates injury progression and contributes to poor patient recovery. Current BBB models primarily include endothelial monocultures and double co-cultures with either astrocytes or pericytes. Such models lack the ability to fully imitate a dynamic brain microenvironment, which is essential for cell-to-cell communication. Additionally, commonly used BBB models often contain immortalized human endothelial cells or animal-derived (rodent, porcine, or bovine) cell cultures that pose translational limitations. This paper describes a novel well-insert-based BBB model containing only primary human cells (brain microvascular endothelial cells, astrocytes, and brain vascular pericytes) enabling the investigation of ischemic brain injury in vitro. The effects of oxygen-glucose deprivation (OGD) on barrier integrity were assessed by passive permeability, transendothelial electrical resistance (TEER) measurements,and direct visualization of hypoxic cells. The presented protocol offers a distinct advantage inmimicking the intercellular environment of the BBB in vivo, serving as a more realistic in vitro BBB model for developing new therapeutic strategies in the setting of ischemic brain injury.

Employing mutants to study the role of a three-membered bacterial consortium in growth promotion of Cajanus cajan

Bacterial consortia, comprising plant growth promoting (PGP) rhizobacteria, are known to outcompete their impacts on plant attributes compared to their individual application. However, tracking of individual bacterial strains post application as consortium, remains challenging. The primary goal of this study was to develop an efficient method of tracking bioinoculants by generating spontaneous mutants of three different bacterial strains in an established consortium, using antibiotic-based screening, followed by their enumeration after application in Cajanus cajan. Mutants were generated for consortium members, viz. Azotobacter chroococcum (A), Priestia megaterium (formerly Bacillus megaterium) (B), and Pseudomonas sp. (P), against streptomycin, kanamycin and rifampicin, respectively. Those mutants having similar growth rates and PGP properties as compared to wild type bacterial strains were selected to test their efficacy in plant growth promotion. Selected mutant strains were applied as mono, dual and triple cultures to C. cajan grown hydroponically. Enumeration of mutant bacterial strains was carried out to check their viability. Bacterial colonization on roots was also analyzed. The application of triple (mutant) inoculants improved plant growth attributes significantly in comparison to mono and dual culture treatments and control. Cell enumeration revealed that the abundance of each bacterial strain increased till the 5th day of treatment. No significant change was observed later in their abundance for any treatment. The triple culture treatment showed greater abundance of bacterial mutant strains in comparison to mono- or dual cultures. To the best of our knowledge, this is the first mutant-based study to have reported the successful tracking and enumeration of bacterial consortium members, post their application in C. cajan.

Core–shell bioprinting of vascularized in vitro liver sinusoid models

In vitro liver models allow the investigation of the cell behavior in disease conditions or in response to changes in the microenvironment. A major challenge in liver tissue engineering is to mimic the tissue-level complexity: besides the selection of suitable biomaterial(s) replacing the extracellular matrix (ECM) and cell sources, the three-dimensional (3D) microarchitecture defined by the fabrication method is a critical factor to achieve functional constructs. In this study, coaxial extrusion-based 3D bioprinting has been applied to develop a liver sinusoid-like model that consists of a core compartment containing pre-vascular structures and a shell compartment containing hepatocytes. The shell ink was composed of alginate and methylcellulose (algMC), dissolved in human fresh frozen plasma. The algMC blend conferred high printing fidelity and stability to the core–shell constructs and the plasma as biologically active component enhanced viability and supported cluster formation and biomarker expression of HepG2 embedded in the shell. For the core, a natural ECM-like ink based on angiogenesis-supporting collagen-fibrin (CF) matrices was developed; the addition of gelatin (G) enabled 3D printing in combination with the plasma-algMC shell ink. Human endothelial cells, laden in the CFG core ink together with human fibroblasts as supportive cells, formed a pre-vascular network in the core in the absence and presence of HepG2 in the shell. The cellular interactions occurring in the triple culture model enhanced the albumin secretion. In conclusion, core–shell bioprinting was shown to be a valuable tool to study cell–cell-interactions and to develop complex tissue-like models.

Investigating the Role of the NLRP3 Inflammasome Pathway in Acute Intestinal Inflammation: Use of THP-1 Knockout Cell Lines in an Advanced Triple Culture Model.

The NLRP3 inflammasome plays an important role in intestinal homeostasis as well as inflammation. However, in vivo studies investigating the role of the NLRP3 inflammasome in inflammatory bowel disease (IBD) report contrasting results, leaving it unclear if the NLRP3 inflammasome augments or attenuates intestinal inflammation. To investigate the role of the NLRP3/caspase-1 pathway in a model of acute intestinal inflammation, we modified a previously established in vitro triple culture model of the healthy and inflamed intestine (Caco-2/HT29-MTX-E12/THP-1). Using THP-1 knockout cell lines, we analyzed how the NLRP3 inflammasome and its downstream enzyme caspase-1 (CASP1) affect inflammatory parameters including barrier integrity and cytotoxicity, as well as gene expression and secretion of pro-inflammatory cytokines and mucus. Furthermore, we investigated differences in inflammation-mediated cytotoxicity towards enterocyte-like (Caco-2) or goblet-like (HT29-MTX-E12) epithelial cells. As a complementary approach, inflammation-related cytotoxicity and gene expression of cytokines was analyzed in intestinal tissue explants from wildtype (WT) and Nlrp3-/- mice. Induction of intestinal inflammation impaired the barrier, caused cytotoxicity, and altered gene expression of pro-inflammatory cytokines and mucins in vitro, while the knockout of NLRP3 and CASP1 in THP 1 cells led to attenuation of these inflammatory parameters. The knockout of CASP1 tended to show a slightly stronger attenuating effect compared to the NLRP3 knockout model. We also found that the inflammation-mediated death of goblet-like cells is NLRP3/caspase-1 dependent. Furthermore, inflammation-related cytotoxicity and upregulation of pro-inflammatory cytokines was present in ileal tissue explants from WT, but not Nlrp3-/- mice. The here presented observations indicate a pro-inflammatory and adverse role of the NLRP3 inflammasome in macrophages during acute intestinal inflammation.

Interlaboratory comparison of an intestinal triple culture to confirm transferability and reproducibility

The development and improvement of advanced intestinal in vitro models has received increasing attention in recent years. While the availability of relevant in vitro models is pivotal to advance the replacement and reduction of animal use in research, their robustness is a crucial determinant for intra- and interlaboratory reproducibility. We have developed a standard protocol to build a triple culture model combining two types of human intestinal epithelial cells (Caco-2, HT29-MTX-E12) and macrophages (THP-1), which was tested for transferability and reproducibility between three laboratories. The epithelial tissue barrier development and triple culture stability were investigated as well as the models’ responses to the non-steroidal anti-inflammatory drug diclofenac in terms of barrier integrity, cytotoxicity, and cytokine release. The results of two partner laboratories were compared to previously established benchmark results and quality criteria. For the epithelial co-cultures, the results were overall highly comparable between the laboratories. The addition of THP-1 cells resulted in increased variability and reduced reproducibility. While good correlation was achieved in several endpoints, others showed substantial response differences between the laboratories. Some variations may be addressed with training or demonstrations, whereas others might be related to fundamental differences in the cell lines introduced during routine cell culture and maintenance. Our results underline the importance of interlaboratory transfer studies using standardised experimental procedures, including defined quality criteria and benchmarks, as well as of training when newly establishing complex in vitro models in laboratories.

Impact of Sr2+ and hypoxia on 3D triple cultures of primary human osteoblasts, osteocytes and osteoclasts

An in vitro bone triple culture involving human primary osteoblasts, osteocytes and osteoclasts enables the investigation of bone healing factors, drugs or biomaterials in a model system for native bone tissue. The present study analyses the impact of Sr2+ as well as hypoxic cultivation (5% O2 content or chemically induced by Co2+) on bone cells. The three cell types were cultivated together in the presence of 100 µM Sr2+, hypoxic conditions or in the presence of 75 µM Co2+. After cultivation the cell types were separated and analysed on mRNA and protein level individually. In response to Sr2+ osteoblasts showed a downregulation of IBSP expression and a stimulation of ALP activity. Osteocyte gene marker expression of PDPN, MEPE, RANKL, OPG, osteocalcin and likewise the amount of secreted osteocalcin was reduced in the presence of Sr2+. Activity of osteoclast-specific enzymes TRAP and CAII was enhanced compared to the Sr2+ free control. Hypoxic conditions induced by both 5% O2 or a Co2+ treatment led to decreased DNA content of all bone cells and downregulated expression of osteoblast markers ALPL and IBSP as well as osteocyte markers PDPN, RANKL and OPG. In addition, Co2+ induced hypoxia decreased gene and protein expression of osteocalcin in osteocytes. In response to the Co2+ treatment, the TRAP gene expression and activity was increased. This study is the first to analyse the effects of Sr2+ or hypoxia on triple cultures with primary human bone cells. The investigated in vitro bone model might be suitable to reduce animal experiments in early stages of biomaterial and drug development.

Pleural Peels Tissue Culture plus Pleural Fluid Culture Help to Improve Culture Rate for Empyema.

Background: Empyema is known as a serious infection, and outcomes of empyema cases remain poor. Pleural fluid culture and blood culture have been reported to give unsatisfactory results. We introduce a novel pleural peels tissue culture during surgery and aim to improve the culture results of empyema. Methods: This was a retrospective study and was obtained from our institute. Patients with stage II or III empyema undergoing video-assisted thoracic surgery decortication from January 2019 to June 2021 were included in the study. Results: There were 239 patients that received a pleural peels tissue culture, a pleural fluid culture, and a blood culture concurrently during the perioperative period. Of these, 153 patients had at least one positive culture and 86 patients showed triple negative culture results. The positive culture rates were 46.9% for pleural peels tissue cultures, 46.0% for pleural fluid cultures, and 10% for blood cultures. The combination of pleural peels tissue culture and pleural fluid culture increased the positive rate to 62.7%. Streptococcus species and Staphylococcus species were the most common pathogens. Conclusion: The combination of pleural peels tissue culture and pleural fluid culture is an effective method to improve the positive culture rate in empyema.

Impact of degradable magnesium implants on osteocytes in single and triple cultures

In vitro triple cultures of human primary osteoblasts, osteocytes and osteoclasts can potentially help to analyze the effect of drugs and degradation products of biomaterials as a model for native bone tissue. In the present study, degradation products of Magnesium (Mg), which has been successfully applied in the biomedical field, were studied with respect to their impact on bone cell morphology and differentiation both in osteocyte single cultures and in the triple culture model. Fluorescence microscopic and gene expression analysis, analysis of osteoclast- and osteoblast-specific enzyme activities as well as osteocalcin protein expression were performed separately for the three cell types after cultivation in triple culture in the presence of extracts, containing 5 and 10 mM Mg2+. All three cell species were viable in the presence of the extracts and did not show morphological changes compared to the Mg-free control. Osteoblasts and osteoclasts did not show significant changes in gene expression of ALPL, BSPII, osteocalcin, TRAP, CTSK and CA2. Likewise on protein level, no significant changes in ALP-, TRAP-, CTSK- and CAII activities were detected. Osteocytes showed a significant downregulation of MEPE, which codes for a protein playing an important role in regulation of phosphate homeostasis by osteocytes. This study is the first to analyze the effects of Mg degradation products on primary osteocytes in vitro, both in single and triple culture. Even if promoting effects on the three examined bone cell species were not found in the applied triple culture setup, it was shown, that Mg degradation products do not interfere with the activity of osteoblasts, osteoclasts and osteocytes in vitro.