Prolonged Culture Period Research Articles

A single gene mutation underpins metabolic adaptation and acquisition of filamentous competence in the emerging fungal pathogen Candida auris.

Filamentous cell growth is a vital property of fungal pathogens. The mechanisms of filamentation in the emerging multidrug-resistant fungal pathogen Candida auris are poorly understood. Here, we show that exposure of C. auris to glycerol triggers a rod-like filamentation-competent (RL-FC) phenotype, which forms elongated filamentous cells after a prolonged culture period. Whole-genome sequencing analysis reveals that all RL-FC isolates harbor a mutation in the C2H2 zinc finger transcription factor-encoding gene GFC1 (Gfc1 variants). Deletion of GFC1 leads to an RL-FC phenotype similar to that observed in Gfc1 variants. We further demonstrate that GFC1 mutation causes enhanced fatty acid β-oxidation metabolism and thereby promotes RL-FC/filamentous growth. This regulation is achieved through a Multiple Carbon source Utilizer (Mcu1)-dependent mechanism. Interestingly, both the evolved RL-FC isolates and the gfc1Δ mutant exhibit an enhanced ability to colonize the skin. Our results reveal that glycerol-mediated GFC1 mutations are beneficial during C. auris skin colonization and infection.

Exploring SSEA3 as an emerging biomarker for assessing the regenerative potential of dental pulp-derived stem cells

BackgroundHuman dental pulp-derived stem cells (hDPSCs) have emerged as a promising source for adult stem cell-based regenerative medicine. Stage-specific embryonic antigen 3 (SSEA3) is a cell surface marker associated with Multilineage-differentiating stress-enduring (Muse) cells, a subpopulation of human bone marrow-derived stem cells (hBMSCs), known for their potent regenerative potential and safety profile. In this study, we investigated the influence of the prolonged culture period and the number of culture passages on the regenerative capacity of hDPSCs and explored the association between SSEA3 expression and their regenerative abilities. MethodshDPSCs were isolated and cultured for up to 20 passages. Cell proliferation, migration, and osteogenic, adipogenic and neurogenic differentiation potential were assessed at passages 5, 10, and 20. Flow cytometry and immunofluorescence were employed to analyze SSEA3 expression. RNA sequencing (RNA-seq) was performed on SSEA3-positive and SSEA3-negative hDPSCs to identify differentially expressed genes and associated pathways. ResultsOur findings demonstrated a progressive decline in hDPSCs proliferation and migration capacity with increasing passage number. Conversely, cell size exhibited a positive correlation with passage number. Early passage hDPSCs displayed superior osteogenic and adipogenic differentiation potential. Notably, SSEA3 expression exhibited a significant negative correlation with passage numbers, reflecting the observed decline in differentiation capacity. RNA-seq analysis revealed distinct transcriptional profiles between SSEA3-positive and SSEA3-negative hDPSCs. SSEA3-positive cells displayed upregulation of genes associated with ectodermal differentiation and downregulation of genes involved in cell adhesion. ConclusionsThis study elucidates the impact of passaging on hDPSC behavior and suggests SSEA3 as a valuable biomarker for evaluating stemness and regenerative potential. SSEA3-positive hDPSCs, functionally analogous to Muse cells, represent a promising cell population for developing targeted regenerative therapies with potentially improved clinical outcomes.

Characterization of diseased primary human hepatocytes in an all-human cell-based triculture system

Liver diseases, including NAFLD, are a growing worldwide health concern. Currently, there is a lack of suitable in vitro models that sustain basic primary human hepatocyte (PHH) morphology and functionality while supporting presentation of disease-associated phenotypic characteristics such as lipid accumulation and inflammasome activation. In TruVivo, an all-human triculture system (hTCS), basic metabolic functions were characterized in PHHs isolated from normal or diseased livers during two-weeks of culture. Decreases in albumin and urea levels and CYP3A4 activity were seen in diseased-origin PHHs compared to normal PHHs along with higher CYP2E1 expression. Positive expression of the macrophage markers CD68 and CD163 were seen in the diseased PHH preparations. Elevated levels of the pro-inflammatory cytokines IL-6 and MCP-1 and the fibrotic markers CK-18 and TGF-β were also measured. Gene expression of FASN, PCK1, and G6PC in the diseased PHHs was decreased compared to the normal PHHs. Further characterization revealed differences in lipogenesis and accumulation of intracellular lipids in normal and diseased PHHs when cultured with oleic acid and high glucose. TruVivo represents a promising new platform to study lipogenic mechanisms in normal and diseased populations due to the preservation of phenotypic differences over a prolonged culture period.

Recent Advances in Maturation of Induced pluripotent Stem Cells-derived Cardiomyocytes

Induced pluripotent stem cells -derived cardiomyocytes (iPSC-CMs) increasingly become an important tool for studying genetic heart diseases, drug screening and heart cell therapy. However, iPSC-CMs differentiated by the traditional ways are immature and the challenge of producing fully matured iPSC-CMs that functionally resemble adult cardiomyocytes persists. Immature iPSC-CMs exhibit characteristics typical of fetal-stage cells, including less developed contractility, electrophysiological properties, and metabolic processes. These limitations restrict their clinical applications, especially in regenerative medicine. Over the past decade, numerous strategies have been developed to enhance the maturation of iPSC-CMs. These include prolonged culture periods, biochemical induction using specific hormones and energy substrates, and the application of biophysical stimuli such as electrical and mechanical stimulation. In addition, advances in biomaterials, such as extracellular matrices and hydrogels, have been crucial in replicating the physiological environment needed to support iPSC-CM development. Together, these methods aim to produce cardiomyocytes that more closely mimic adult heart cells in structure and function. This review explores recent progress in optimizing the maturation of iPSC-CMs and highlights their potential impact on future applications in cell therapy and heart disease modeling.

Novel methodologies for host-microbe interactions and microbiome-targeted therapeutics in 3D organotypic skin models

BackgroundFollowing descriptive studies on skin microbiota in health and disease, mechanistic studies on the interplay between skin and microbes are on the rise, for which experimental models are in great demand. Here, we present a novel methodology for microbial colonization of organotypic skin and analysis thereof.ResultsAn inoculation device ensured a standardized application area on the stratum corneum and a homogenous distribution of bacteria, while preventing infection of the basolateral culture medium even during prolonged culture periods for up to 2 weeks at a specific culture temperature and humidity. Hereby, host-microbe interactions and antibiotic interventions could be studied, revealing diverse host responses to various skin-related bacteria and pathogens.ConclusionsOur methodology is easily transferable to a wide variety of organotypic skin or mucosal models and different microbes at every cell culture facility at low costs. We envision that this study will kick-start skin microbiome studies using human organotypic skin cultures, providing a powerful alternative to experimental animal models in pre-clinical research.Dkr_4cuqp92zgpqQmj5SV7Video

Oleic acid improves the conidial production and quality of Metarhizium rileyi as a biocontrol agent

ABSTRACT Metarhizium rileyi is a biological agent used in the control of various pests, particularly noctuid insects. However, this fungal species is slow-growing, resulting in a prolonged culture period and limiting its commercial production. In this study, 0.3% (w/w) oleic acid significantly enhanced the rates of hyphal growth, conidial production, and conidial germination by 23%, 9%, and 10%, respectively. The expression of genes related to microbial growth and development (MrPbs2, MrMsn2, MrSwi6, MrNsdD, Mrap1 and MrSte12) was significantly increased in the presence of oleic acid. The M. rileyi conidial production and its effects on the mortality of Spodoptera frugiperda were investigated using rice-vegetable oil solid medium (RVOSM) and its substitute Sabouraud maltose agar plus yeast extract (SMAY) plates containing 0.03% oleic acid. The fungi retained their toxicity to S. frugiperda. Therefore, oleic acid can improve the growth potential of fungi, and RVOSM can be used as a substrate for conidia production for the application of M. rileyi as a biocontrol agent.

Optimal Dietary Protein Requirement of Subadult Australian Hybrid Abalone (Haliotis rubra × Haliotis laevigata) at Different Rearing Temperatures

Australian abalone aquaculture is characterised by a prolonged culture period and slow and variable growth, and abalone is cultured in fluctuating water temperatures ranging between 10 and 25°C with distinct seasons. Temperature is a crucial environmental factor influencing abalone’s physiology and energetics, leading to a change in nutritional requirements. However, feeds are generally formulated to match the nutritional requirements at their optimal temperature. Hence, there is a need to optimise dietary protein levels to match temperature-specific requirements during extreme conditions (winter and summer). Given this, a growth trial evaluating five experimental feeds consisting of graded protein inclusion levels (320, 350, 380, 410, and 440 g·kg−1) was conducted on subadult hybrid abalone (Haliotis rubra × H. laevigata) at three different temperatures reflecting winter (12°C), summer (22°C), and the annual average water temperature (17°C) for 143 days. At lower water temperature (12°C), there was a marginal improvement in growth performance as dietary protein levels increased from 320 to 440 g·kg−1. However, at higher water temperatures (when the culture water temperature is above 17°C), there was a significant improvement in growth performance as dietary protein levels increased from 320 to 440 g·kg−1 as evidenced by an improved weight gain, specific growth rate, and feed conversion ratio. Furthermore, increasing dietary protein levels did not compromise the nutritional quality of the abalone tissue at all three tested temperatures. Therefore, during periods of higher water temperatures, feeding Australian hybrid abalone with a relatively high dietary protein level (410 g·kg−1) is expected to result in improved growth, shorter culture duration, and profit maximisation.

Single Nucleotide Polymorphism (SNP) Arrays and Their Sensitivity for Detection of Genetic Changes in Human Pluripotent Stem Cell Cultures.

Human pluripotent stem cells (hPSCs) can be grown in culture indefinitely, making them a valuable tool for use in basic biology, disease modeling, and regenerative medicine. However, over prolonged periods in culture, hPSCs tend to acquire genomic aberrations that confer growth advantages, similar to those seen in some cancers. Monitoring the genomic stability of cultured hPSCs is critical to ensuring their efficacy and safety as a therapeutic tool. Most commonly employed methods for monitoring of hPSC genomes are cytogenetic methods, such as G-banding. Nonetheless, such methods have limited resolution and sensitivity for detecting mosaicism. Single nucleotide polymorphism (SNP) array platforms are a potential alternative that could improve detection of abnormalities. Here, we outline protocols for SNP array whole-genome screening of hPSCs. Moreover, we detail the procedure for assessing the SNP array's sensitivity in detecting low-level mosaic copy-number changes. We show that mosaicism can be confidently identified in samples only once they contain 20% variants, although samples containing 10% variants typically display enough variation to warrant further investigation and confirmation, for example by using a more sensitive targeted method. Finally, we highlight the advantages and limitations of SNP arrays, including a cost comparison of SNP arrays versus other commonly employed methods for detection of genetic changes in hPSC cultures. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: DNA sample preparation for SNP arrays Basic Protocol 2: SNP array hybridization, washing, and scanning Basic Protocol 3: SNP array data analysis Support Protocol: Assessment of SNP array sensitivity for detection of mosaicism.

Improved anti-tumor T cell generation using rapidly differentiated dendritic cells

Abstract After allogeneic stem cell transplantation, minor histocompatibility antigens (MiHAs), Wilms-Tumor 1 (WT1) antigen, and other peptides presented by host hematologic cancer (HC) cells can lead to the in vivo expansion of donor T cells crucial for the graft-versus-leukemia effect. While the specific ex vivo production of such leukemia-specific T cells represents an interesting treatment approach, the generation of these antigen-specific donor T cells represents a major challenge. We have developed a protocol enabling the infusion of monocyte derived dendritic cell (DC)-induced ex vivo expanded T cells specific for MiHAs, WT1 and other peptides that are preferentially expressed on hematologic cells for the treatment of relapsed HC patients. While it provides interesting results, our 42-day expansion protocol is costly and requires prolonged culture periods during which patient with HC could deteriorate rapidly. To accelerate T cell production, we compared DCs generated in 3 days (DC3) vs 9 days (DC9) using standard maturation cocktail and toll-like receptor agonist. DC3 showed small differences in expression of maturation/activation markers and a unique gene signature vs DC9. Importantly, the frequency of WT1-specific CD8 T cells, primed by DC3, was slightly higher than that in DC9 primed cultures. Upon restimulation, these DC3-primed-WT1-specific T cells expressed higher amounts of IFNγ, TNFα, and CD107a degranulation marker, and were cytotoxic against WT1-expressing autologous blast cells. Together, these data show that matured DC3 have a strong capacity to prime and expand functional anti-tumor antigen-specific CD8 T cells and thus offer most interesting features for ex vivo expansion of T cells for cancer immunotherapy.

Isolation of mouse pancreatic islet Procr+ progenitors and long-term expansion of islet organoids in vitro.

Insulin production is required for glucose homeostasis. Pancreatic islet β cells are the only cells that produce insulin in humans; however, generation of functional β cells in vitro from embryonic or adult tissues has been challenging. Here, we describe isolation of pancreatic islet progenitors from adult mice, which enables the efficient generation and long-term expansion of functional islet organoids in vitro. This protocol starts with purification of protein C receptor (Procr)-expressing islet progenitors. Coculture with endothelial cells generates islet organoids in vitro that can be expanded by passage. Functional maturation is achieved as a consequence of a prolonged culture period and cyclic glucose stimulation. Primary islet organoids form in 7-10 days. Subsequently, each passage takes 1 week, with the final maturation step requiring 3 weeks of additional culture. The resulting organoids are predominantly composed of β cells but also contain small proportions of α, δ and pancreatic polypeptide cells. The organoids sense glucose and secrete insulin. This approach thus provides a strategy for β cell generation in vitro and an organoid system to study islet regeneration and diseases.

Using a micro-physiological system to prolong the preservation of ex vivo lung tissue

Abstract Current in vitro and in vivo disease models have been reported to lack sufficient translation to human. Precision-Cut Lung Slices (PCLS) are viable sections of lung tissue and have been described to be a translational model for the ex vivo assessment of pharmacological and toxicological compounds. In most studies PCLS were cultured under static conditions. These lung sections, however, suffer from the limited viability. Here we present a novel modular microphysiological system (MPS) to prolong the cultivation of ex vivo lung tissue. A tailored MPS setup was designed using the PDMS free modular plug&play MPS construction kit. PCLS from mice were cultivated for up to one week under static versus perfused conditions. Using the MPS technology enabled a prolonged culture period with improved viability as shown by lowered lactate dehydrogenase release and improved membrane integrity. Using this technology might allow us to use PCLS for longer culture periods such as e.g. repeated dose toxicity or pharmacology studies.

Characterization of stable hypoxia-preconditioned dental pulp stem cells compared with mobilized dental pulp stem cells for application for pulp regenerative therapy

BackgroundDental pulp stem cells (DPSCs) have been developed as a potential source of mesenchymal stem cells (MSCs) for regeneration of dental pulp and other tissues. However, further strategies to isolate highly functional DPSCs beyond the colony-forming methods are required. We have demonstrated the safety and efficacy of DPSCs isolated by G-CSF-induced mobilization and cultured under normoxia (mobilized DPSCs, MDPSCs) for pulp regeneration. The device for isolation of MDPSCs, however, is not cost-effective and requires a prolonged cell culture period. It is well known that MSCs cultured under hypoxic-preconditions improved MSC proliferation activity and stemness. Therefore, in this investigation, we attempted to improve the clinical utility of DPSCs by hypoxia-preconditioned DPSCs (hpDPSCs) compared with MDPSCs to improve the potential clinical utility for pulp regeneration in endodontic dentistry.MethodsColony-forming DPSCs were isolated and preconditioned with hypoxia in a stable closed cultured system and compared with MDPSCs isolated from the individual dog teeth. We examined the proliferation rate, migration potential, anti-apoptotic activity, and gene expression of the stem cell markers and angiogenic/neurotrophic factors. Trophic effects of the conditioned medium (CM) were also evaluated. In addition, the expression of immunomodulatory molecules upon stimulation with IFN-γ was investigated. The pulp regenerative potential and transplantation safety of hpDPSCs were further assessed in pulpectomized teeth in dogs by histological and immunohistochemical analyses and by chemistry of the blood and urine tests.ResultshpDPSCs demonstrated higher proliferation rate and expression of a major regulator of oxygen homeostasis, HIF-1α, and a stem cell marker, CXCR-4. The direct migratory activity of hpDPSCs in response to G-CSF was significantly higher than MDPSCs. The CM of hpDPSCs stimulated neurite extension. However, there were no changes in angiogenic, migration, and anti-apoptotic activities compared with the CM of MDPSCs. The expression of immunomodulatory gene, PTGE was significantly upregulated by IFN gamma in hpDPSCs compared with MDPSCs. However, no difference in nitric oxide was observed. The regenerated pulp tissue was quantitatively and qualitatively similar in hpDPSC transplants compared with MDPSC transplants in dog teeth. There was no evidence of toxicity or adverse events of the hpDPSC transplantation.ConclusionsThese results demonstrated that the efficacy of hpDPSCs for pulp regeneration was identical, although hpDPSCs improved stem cell properties compared to MDPSCs, suggesting their potential clinical utility for pulp regeneration.

Double network laminarin-boronic/alginate dynamic bioink for 3D bioprinting cell-laden constructs

The design of dynamically crosslinked hydrogel bioinks for three-dimensional (3D) bioprinting is emerging as a valuable strategy to advance the fabrication of mechanically tuneable cell-laden constructs for 3D in vitro disease modelling and tissue engineering applications. Herein, a dynamic bioink comprising boronic acid-functionalised laminarin and alginate is explored for bioprinting 3D constructs under physiologically relevant conditions. The formulated bioink takes advantage of a double crosslinked network that combines covalent but reversible boronate ester bonds and ionic gelation via divalent cations. Moreover, it exhibits suitable rheological properties and improved mechanical features owing to its modular crosslinking chemistry, yielding stable constructs with user-programmable architecture. We explored such dynamic bioink as a supporting matrix for different cell classes, namely osteoblast precursors, fibroblasts and breast cancer cells. The resulting cell-laden bioprinted hydrogels display a homogeneous cell distribution post-printing and exceptional cell viability (>90%) that can be maintained for prolonged time periods in culture (14 days) for all cell lines. This simple and chemically versatile approach is envisaged to accelerate the development of multifunctional bioinks and contribute towards the fabrication of biomimetic 3D scaffolds with applicability in a wide range of predictive or exploratory biomedical platforms.

Microelectrode Array based Functional Testing of Pancreatic Islet Cells.

Electrophysiological techniques to characterize the functionality of islets of Langerhans have been limited to short-term, one-time recordings such as a patch clamp recording. We describe the use of microelectrode arrays (MEAs) to better understand the electrophysiology of dissociated islet cells in response to glucose in a real-time, non-invasive method over prolonged culture periods. Human islets were dissociated into singular cells and seeded onto MEA, which were cultured for up to 7 days. Immunofluorescent imaging revealed that several cellular subtypes of islets; β, δ, and γ cells were present after dissociation. At days 1, 3, 5, and 7 of culture, MEA recordings captured higher electrical activities of islet cells under 16.7 mM glucose (high glucose) than 1.1 mM glucose (low glucose) conditions. The fraction of the plateau phase (FOPP), which is the fraction of time with spiking activity recorded using the MEA, consistently showed distinguishably greater percentages of spiking activity with high glucose compared to the low glucose for all culture days. In parallel, glucose stimulated insulin secretion was measured revealing a diminished insulin response after day 3 of culture. Additionally, MEA spiking profiles were similar to the time course of insulin response when glucose concentration is switched from 1.1 to 16.7 mM. Our analyses suggest that extracellular recordings of dissociated islet cells using MEA is an effective approach to rapidly assess islet functionality, and could supplement standard assays such as glucose stimulate insulin response.

Meiotic Maturation and Function of Lipidome in Porcine Oocytes That Have Completed Their In Vivo or In Vitro Growth Phase after a Prolonged Culture Period

The kinetics of meiotic chromatin modifications and the function of lipidome in the oocytes that have completed their in vivo or in vitro growth phase after a prolonged culture period have been analyzed in order to improve the efficiency of a technology for extracorporeal maturation of porcine donor-derived oocytes. It is revealed that a prolonged period (up to 50 h) of culturing in the Sage Cleavage medium (CooperSurgical, United States) with the addition of 5% Serum Protein Substitute supplement (Cooper Surgical, United States) and 10 IU human chorionic gonadotropin can provoke destruction of chromatin of BCB+-positive and BCB–-negative oocytes (76 vs. 50%, P < 0.01). The age-associated porcine oocyte processes are usually followed by changes in lipid droplet morphology, since the granules tend to transform into clusters. A high percent of BCB–-negative oocytes containing normal chromatin in metaphase 2 can indicate the capability of oocytes that have not completed their in vivo growth phases to complete it in vitro with the subsequent meiotic maturation. The produced data may be used to model the maturation systems for porcine female gametes that have not completed their in vivo growth phases.

Alveolar Extracellular Matrix Hydrogels Facilitate Proliferation of Induced Pluripotent Stem Cell-Derived Alveolar Epithelial Spheroids

Background & Aim Type 2 alveolar epithelial cells (AEC2s) are adult cells in the human lung that have a critical role in lung homeostasis. In addition to secreting surfactant that is necessary to prevent alveolar collapse, AEC2s are the facultative progenitors of the alveolus. Specifically, in response to injury AEC2s proliferate to replenish AEC2s and type 1 alveolar epithelial cells (AEC1s). However, despite the critical role of AEC2s in lung physiology and injury response, an inability to isolate an expandable culture of AEC2 in vitro has impeded a full understanding of AEC2 biology. Recently, a pure and expandable culture of induced-AEC2s (iAEC2s) spheroids (i.e. alveolospheres) has been derived using a directed differentiation method from induced pluripotent stem cells (iPSCs). These iAEC2s spheroids are amenable to prolonged culture periods, possess mature markers and functionality (e.g. surfactant secretion) of in vivo AEC2s, and can recapitulate AEC2 dysfunction. However, initial studies of iAEC2s were performed in Matrigel, an extracellular matrix (ECM) derived from a tumorigenic mouse cell line, and it is unknown how iAEC2s respond to a physiologically relevant lung matrix. Methods, Results & Conclusion Here, we employ a novel and innovative culture method to determine how iAEC2s respond to human alveolar ECM. Decellularized lungs from adult patients with a healthy lung history were used to produce 3-dimensional hydrogels from alveolar enriched ECM (aECM) for the culture of iAEC2s. Our preliminary results provide evidence that iAEC2s maintain alveolosphere formation and efficiently proliferate in aECM hydrogels in an aECM protein concentration dependent manner. Furthermore, gene expression analysis suggests that aECM promotes lamellar body associated AEC2 maturation markers (i.e. SFTPC, LAMP3, ABCA3) in iAEC2s. Importantly, the ability to culture iAEC2s in a physiologically relevant lung matrix could provide a unique model to determine the effects of normal and diseased lung ECM (e.g. idiopathic pulmonary fibrosis, chronic obstructive pulmonary disease) on AEC2 behavior.

Trauma induced tissue survival in vitro with a muscle-biomaterial based osteogenic organoid system: a proof of concept study

BackgroundThe translation from animal research into the clinical environment remains problematic, as animal systems do not adequately replicate the human in vivo environment. Bioreactors have emerged as a good alternative that can reproduce part of the human in vivo processes at an in vitro level. However, in vitro bone formation platforms primarily utilize stem cells only, with tissue based in vitro systems remaining poorly investigated. As such, the present pilot study explored the tissue behavior and cell survival capability within a new in vitro skeletal muscle tissue-based biomaterial organoid bioreactor system to maximize future bone tissue engineering prospects.ResultsThree dimensional printed β-tricalcium phosphate/hydroxyapatite devices were either wrapped in a sheet of rat muscle tissue or first implanted in a heterotopic muscle pouch that was then excised and cultured in vitro for up to 30 days. Devices wrapped in muscle tissue showed cell death by day 15. Contrarily, devices in muscle pouches showed angiogenic and limited osteogenic gene expression tendencies with consistent TGF-ß1, COL4A1, VEGF-A, RUNX-2, and BMP-2 up-regulation, respectively. Histologically, muscle tissue degradation and fibrin release was seen being absorbed by devices acting possibly as a support for new tissue formation in the bioceramic scaffold that supports progenitor stem cell osteogenic differentiation.ConclusionsThese results therefore demonstrate that the skeletal muscle pouch-based biomaterial culturing system can support tissue survival over a prolonged culture period and represents a novel organoid tissue model that with further adjustments could generate bone tissue for direct clinical transplantations.

PB2102 A SAME DAY ASSAY USING CYTOCHROME C RELEASE DETERMINES SENSITIVITY TO COOPERATIVE BCL‐2 AND MCL‐1 TARGETING IN MULTIPLE MYELOMA CELL LINES AND PRIMARY SAMPLES

Background:Multiple myeloma is an incurable haematological malignancy with remarkable clonal and genetic heterogeneity. Although both MCL‐1 and BCL‐2 are necessary for myeloma survival, most myelomas are dependent on MCL‐1 for survival such that BCL‐2 inhibition alone only yields significant cytotoxicty in a minority and cooperative inhibition of both proteins is required. Knowing which patients will respond can be challenging since cytotoxicty assessment of primary patient samples is significantly hampered by the lack of longevity of those cells in culture ex vivo.Aims:We aimed to study whether a short term flow cytometric assay could be used to determine cytotoxicity in myeloma cell lines and primary samples after cooperative inhibition of BCL‐2 and MCL‐1 by the BH3 mimetics (venetoclax and S63845).MethodsMyeloma cell lines and patient samples were treated with venetoclax and S63845 alone and in combinations. Sub‐toxic doses of drugs were determined for each cell line. Samples were fixed after 4 hours drug incubation thus precluding the need to keep those cells alive in culture for prolonged periods and then assayed for cytochrome c release using flow cytometry. The Bliss independence model was used to demonstrate synergy.Results:The cytochrome C release assay demonstrated that the combination of venetoclax and S63845 was synergistic as compared to controls and single agent treatment in all six cell lines tested (Combination index values 7.8‐80). All three cyclin D2 translocated cell lines were particularly sensitive to the combination and synergy was p53 independent. Furthermore, the same day assay was used on CD138‐selected primary myeloma cells also demonstrating effective cooperative targeting in patient samples.Summary/Conclusion:The attractiveness in this assay lies in its ability as a predictor of apoptotic response to chemotherapy and molecular targeting agents providing rapid results. Here, we have demonstrated for the first time in myeloma that using the cytochrome C release assay we can quickly predict apoptosis in cell lines. Importantly we are now demonstrating that this assay can also be used in primary samples. This can set the steps for an individualised assay to determine patient responses to targeted molecular therapies.

Development of an alternating tangential flow (ATF) perfusion-based transient gene expression (TGE) bioprocess for universal influenza vaccine.

An alternating tangential flow (ATF) perfusion-based transient gene expression (TGE) bioprocess has been developed using human embryonic kidney (HEK) 293 cells to produce H1-ss-np, a promising candidate for a universal influenza vaccine. Two major adjustments were taken to improve the process: (1) eliminate the interference of microbubbles during gene transfection; and (2) utilize an ATF perfusion system for a prolonged culture period. As a result, a closed-operation 9-days ATF perfusion-based TGE bioprocess was developed. The TGE bioprocess showed continuous cell growth with high cell viability and prolonged cellular productivity that achieved recombinant product level of ~270 mg/L which was more than two times that of 4-days base-line TGE bioprocess. In addition, the consumables cost per milligram for ATF perfusion-based TGE bioprocess was ~70% lower than that of the base-line TGE bioprocess suggesting high cost savings potential in vaccine manufacturing. Based on the lower contamination risk, higher productivity, and cost efficiency, the ATF perfusion-based TGE bioprocess can likely provide potential benefits to many future applications in vaccine and drug manufacturing.

Rates of antibiotic resistance/sensitivity in bacterial cultures of hidradenitis suppurativa patients.

Antibiotic (AB) treatment is one of the first steps in the management of hidradenitis suppurativa (HS). Bacteria, in HS patients, may play a double role, as triggering factors of inflammatory reactions and/or agents of infection. The aims of this study are as follows: (i) to assess prevalence and AB resistance of bacterial growths in HS patients (ii) assessment of the clinical relevance of obtained data in guiding the selection of the most effective AB therapy. Purulent material from 137 skin lesions of HS patients was collected with swabs. Bacterial flora and AB sensitivity were determined using microbiological cultures for aerobic and anaerobic bacteria. A total of 114 samples resulted positive for bacteria. Sample was collected from the axillae, groin and perianal areas. A total of 163 single bacterial growths were observed; 55% were Gram-positive and 44% were Gram-negative. Among them, 18.4% were anaerobic. The most frequent bacterial families included enterobacteriaceae (30.7%), Staphylococcus (25.2%) and Streptococcus (14.1%). The most frequent genus or species were proteus spp. (13.5%) and Escherichia coli (9.8%). The prevalence of AB resistance observed was clindamycin 65.7%, rifampicin 69.3%, penicillin 70.0%, ciprofloxacin 74%, tetracycline 84.7% and erythromycin 89.0%. A limitation of the study is represented the short culture period adopted which may have impaired the isolation of anaerobes. Bacterial growth in HS patients has shown a high level of resistance to ABs, including rifampicin, clindamycin and tetracyclines, cited as an empiric choice in HS therapeutic guidelines. A targeted and specific AB therapy, driven by microbiological evaluations with prolonged culture periods, seems more appropriate than empiric, generic, non-specific, therapeutic approaches. Current knowledge regarding HS bacterial AB resistance should be considered in the update of current therapeutic guidelines for HS.