Native Phenotype Research Articles

A 3D in vitro model of biphasic calcium phosphate (BCP) scaffold combined with human osteoblasts, osteoclasts, and endothelial cells as a platform to mimic the oral microenvironment for tissue regeneration

Objectives: This study aimed to develop an innovative 3D in vitro model based on the biphasic calcium phosphate (BCP) scaffold combined with human osteoblasts (hOBs), osteoclasts (hOCs), and endothelial cells to evaluate its effects on bone and vascular cells behavior. Methods: To this end, an optimized mixture of hydroxyapatite (HA) and β-tricalcium phosphate (TCP) with a weight ratio of 30/70 was employed to develop a BCP scaffold using the computer-aided design (CAD) approach. The BCP scaffold was combined with primary cultures of hOBs, hOCs and human umbilical vein endothelial cells (HUVECs). Results: Morphometric analyses using scanning electron microscopy (SEM) and X-ray micro-computed tomography, along with biomechanical testing, revealed that BCP scaffold exhibited a regular 3D structure with large interconnected internal pores (700 µm) and high mechanical strength. In terms of biological behavior, after 14 days of tri-culture with hOBs, hMCs and HUVECs, SEM, immunofluorescence, and histological analyses showed that all cell types were viable and adhered well to the entire surface of the scaffold. Interestingly, SEM and energy-dispersive X-ray spectroscopy analyses also revealed on the BCP scaffold the presence of mineralized matrix crystals of Ca, P, O and C within a tissue-like cell layer produced by the interaction of the three cell types. Conclusions: Data confirmed the high performance of the BCP scaffold through biomechanical studies. Notably, for the first time, this study demonstrated the feasibility of combining BCP scaffold with hOBs, hOCs, and HUVEC, which remained viable and maintained their native phenotypes, creating also tissue-like cell layer. Clinical significanceAlthough further investigation is needed, these results underscore the potential to develop a 3D in vitro model that mimics the oral microenvironment, which could be valuable for BTE approaches in in vivo studies.

Berberine potentiates liver inflammation and fibrosis in the PI*Z hAAT transgenic murine model.

Alpha-1 antitrypsin deficiency (AATD) is an inherited disease, the common variant caused by a Pi*Z mutation in the SERPINA1 gene. Pi*Z AAT increases the risk of pulmonary emphysema and liver disease. Berberine (BBR) is a nature dietary supplement and herbal remedy. Emerging evidence revealed that BBR has remarkable liver-protective properties against various liver diseases. In the present study, we investigated the therapeutic effects and toxicities of BBR in Pi*Z hepatocytes and Pi*Z transgenic mice. Huh7.5 and Huh7.5Z (which carries the Pi*Z mutation) cells were treated with different concentrations of BBR for 48 hours. MTT was performed for cell viability assay. Intracellular AAT levels were evaluated by western blot. In vivo studies were carried out in wild type, native phenotype AAT (Pi*M), and Pi*Z AAT transgenic mice. Mice were treated with 50 mg/kg/day of BBR or solvent only by oral administration for 30 days. Western blot and liver histopathological examinations were performed to evaluate therapeutic benefits and liver toxicity of BBR. BBR reduced intracellular AAT levels in Huh7.5Z cells, meanwhile, no Pi*Z-specific toxicity was observed. However, BBR did not reduce liver AAT load but significantly potentiated liver inflammation and fibrosis accompanying the activation of unfolded protein response and mTOR in Pi*Z mice, but not in wild type and Pi*M mice. BBR exacerbated liver inflammation and fibrosis specifically in Pi*Z mice. This adverse effect may be associated with the activation of unfolded protein response and mTOR. This study implicates that BBR should be avoided by AATD patients.

Formation and culture of cell spheroids by using magnetic nanostructures resembling a crown of thorns

In contrast to traditional two-dimensional cell-culture conditions, three-dimensional (3D) cell-culture models closely mimic complexin vivoconditions. However, constructing 3D cell culture models still faces challenges. In this paper, by using micro/nano fabrication method, including lithography, deposition, etching, and lift-off, we designed magnetic nanostructures resembling a crown of thorns. This magnetic crown of thorns (MCT) nanostructure enables the isolation of cells that have endocytosed magnetic particles. To assess the utility of this nanostructure, we used high-flux acquisition of Jurkat cells, an acute-leukemia cell line exhibiting the native phenotype, as an example. The novel structure enabled Jurkat cells to form spheroids within just 30 min by leveraging mild magnetic forces to bring together endocytosed magnetic particles. The size, volume, and arrangement of these spheroids were precisely regulated by the dimensions of the MCT nanostructure and the array configuration. The resulting magnetic cell clusters were uniform in size and reached saturation after 1400 s. Notably, these cell clusters could be easily separated from the MCT nanostructure through enzymatic digestion while maintaining their integrity. These clusters displayed a strong proliferation rate and survival capabilities, lasting for an impressive 96 h. Compared with existing 3D cell-culture models, the approach presented in this study offers the advantage of rapid formation of uniform spheroids that can mimicin vivomicroenvironments. These findings underscore the high potential of the MCT in cell-culture models and magnetic tissue enginerring.

Environmental effects on genetic variance are likely to constrain adaptation in novel environments.

Adaptive plasticity allows populations to cope with environmental variation but is expected to fail as conditions become unfamiliar. In novel conditions, populations may instead rely on rapid adaptation to increase fitness and avoid extinction. Adaptation should be fastest when both plasticity and selection occur in directions of the multivariate phenotype that contain abundant genetic variation. However, tests of this prediction from field experiments are rare. Here, we quantify how additive genetic variance in a multivariate phenotype changes across an elevational gradient, and test whether plasticity and selection align with genetic variation. We do so using two closely related, but ecologically distinct, sister species of Sicilian daisy (Senecio, Asteraceae) adapted to high and low elevations on Mt. Etna. Using a quantitative genetic breeding design, we generated and then reciprocally planted c. 19,000 seeds of both species, across an elevational gradient spanning each species' native elevation, and then quantified mortality and five leaf traits of emergent seedlings. We found that genetic variance in leaf traits changed more across elevations than between species. The high-elevation species at novel lower elevations showed changes in the distribution of genetic variance among the leaf traits, which reduced the amount of genetic variance in the directions of selection and the native phenotype. By contrast, the low-elevation species mainly showed changes in the amount of genetic variance at the novel high elevation, and genetic variance was concentrated in the direction of the native phenotype. For both species, leaf trait plasticity across elevations was in a direction of the multivariate phenotype that contained a moderate amount of genetic variance. Together, these data suggest that where plasticity is adaptive, selection on genetic variance for an initially plastic response could promote adaptation. However, large environmental effects on genetic variance are likely to reduce adaptive potential in novel environments.

Self-Assembled Fibrinogen Scaffolds Support Cocultivation of Human Dermal Fibroblasts and HaCaT Keratinocytes.

Self-assembled fibrinogen scaffolds are highly attractive biomaterials to mimic native blood clots. To explore their potential for wound healing, we studied the interaction of cocultures of human dermal fibroblasts (HDFs) and HaCaT keratinocytes with nanofibrous, planar, and physisorbed fibrinogen. Cell viability analysis indicated that the growth of HDFs and HaCaTs was supported by all fibrinogen topographies until 14 days, either in mono- or coculture. Using scanning electron microscopy and cytoskeletal staining, we observed that the native morphology of both cell types was preserved on all topographies. Expression of the marker proteins vimentin and cytokeratin-14 showed that the native phenotype of fibroblasts and undifferentiated keratinocytes, respectively, was maintained. HDFs displayed their characteristic wound healing phenotype, characterized by expression of fibronectin. Finally, to mimic the multilayered microenvironment of skin, we established successive cocultures of both cells, for which we found consistently high metabolic activities. SEM analysis revealed that HaCaTs arranged into a confluent top layer after 14 days, while fluorescent labeling confirmed the presence of both cells in the layered structure after 6 days. In conclusion, all fibrinogen topographies successfully supported the cocultivation of fibroblasts and keratinocytes, with fibrinogen nanofibers being particularly attractive for skin regeneration due to their biomimetic porous architecture and the technical possibility to be detached from an underlying substrate.

Induced pluripotent stem cells reprogramming overcomes technical limitations for highly pigmented adult melanocyte amplification and integration in 3D skin model.

Understanding pigmentation regulations taking into account the original skin color type is important to address pigmentary disorders. Biological models including adult melanocytes from different phenotypes allow to perform fine-tuned explorative studies and support discovery of treatments adapted to populations' skin color. However, technical challenges arise when trying to not only isolate but also amplify melanocytes from highly pigmented adult skin. To bypass the initial isolation and growth of cutaneous melanocytes, we harvested and expanded fibroblasts from light and dark skin donors and reprogrammed them into iPSC, which were then differentiated into melanocytes. The resulting melanocyte populations displayed high purity, genomic stability, and strong proliferative capacity, the latter being a critical parameter for dark skin cells. The iPSC-derived melanocyte strains expressed lineage-specific markers and could be successfully integrated into reconstructed skin equivalent models, revealing pigmentation status according to the native phenotype. In both monolayer cultures and 3D skin models, the induced melanocytes demonstrated responsiveness to promelanogenic stimuli. The data demonstrate that the iPSC-derived melanocytes with high proliferative capacity maintain their pigmentation genotype and phenotypic properties up to a proper integration into 3D skin equivalents, even for highly pigmented cells.

A Novel In Vitro Disease Model for Systemic Sclerosis Using Polyisocyanide Hydrogels

AbstractSystemic sclerosis (SSc) is a rare autoimmune disease with limited treatment options that is characterized by fibrosis in various organs. To screen the effectiveness of new therapies, there is an urgent need for reliable in vitro models. Key is that diseased cells' characteristics are maintained, which is challenging in currently used setups. In this study, an in vitro 3D culture system is described using the biocompatible polyisocyanide (PIC‐RGD) hydrogel and SSc patient‐derived fibroblasts from affected (lesional cells) and from healthy‐skin (healthy cells). In contrast to the standard collagen‐coated 2D cultures, the cells in the 3D PIC‐RGD gels maintain the native phenotype and functionality of the primary cells. The functionality of the model is studied in the presence of the fibrosis stimulator transforming growth factor β1 (TGFβ1) and the suppressor tumor necrosis factor (TNFα). In this study, it is observed that lesional cells have a stronger fibrotic character with increased contraction, proliferation, and expression of collagen, and myofibroblast markers α‐smooth muscle actin and fibroblast activation protein. The high tunability of the hydrogel, which can maintain the native functionality of fibroblasts in in vitro cultures, delivers a crucial step in developing these materials into an effective tool for personalized medicine approaches of SSc patients.

Formulation and evaluation of a bioink composed of alginate, gelatin, and nanocellulose for meniscal tissue engineering.

1The necessity to preserve meniscal function prompts the research and development of novel treatment options, like three-dimensional (3D) bioprinting. However, bioinks for meniscal 3D bioprinting have not been extensively explored. Therefore, in this study, a bioink composed of alginate, gelatin, and carboxymethylated cellulose nanocrystal (CCNC) was formulated and evaluated. Firstly, bioinks with varying concentrations of the aforementioned components were subjected to rheological analysis (amplitude sweep test, temperature sweep test, and rotation). The optimal bioink formulation of 4.0% gelatin, 0.75% alginate, and 1.4% CCNC dissolved in 4.6% D-mannitol was further used for printing accuracy analysis, followed by 3D bioprinting with normal human knee articular chondrocytes (NHAC-kn). The encapsulated cells' viability was > 98%, and collagen II expression was stimulated by the bioink. The formulated bioink is printable, stable under cell culture conditions, biocompatible, and able to maintain the native phenotype of chondrocytes. Aside from meniscal tissue bioprinting, it is believed that this bioink could serve as a basis for the development of bioinks for various tissues.

Quantifying Cell-Derived Changes in Collagen Synthesis, Alignment, and Mechanics in a 3D Connective Tissue Model.

Dysregulation of extracellular matrix (ECM) synthesis, organization, and mechanics are hallmark features of diseases like fibrosis and cancer. However, most in vitro models fail to recapitulate the three‐dimensional (3D) multi‐scale hierarchical architecture of collagen‐rich tissues and as a result, are unable to mirror native or disease phenotypes. Herein, using primary human fibroblasts seeded into custom fabricated 3D non‐adhesive agarose molds, a novel strategy is proposed to direct the morphogenesis of engineered 3D ring‐shaped tissue constructs with tensile and histological properties that recapitulate key features of fibrous connective tissue. To characterize the shift from monodispersed cells to a highly‐aligned, collagen‐rich matrix, a multi‐modal approach integrating histology, multiphoton second‐harmonic generation, and electron microscopy is employed. Structural changes in collagen synthesis and alignment are then mapped to functional differences in tissue mechanics and total collagen content. Due to the absence of an exogenously added scaffolding material, this model enables the direct quantification of cell‐derived changes in 3D matrix synthesis, alignment, and mechanics in response to the addition or removal of relevant biomolecular perturbations. To illustrate this, the effects of nutrient composition, fetal bovine serum, rho‐kinase inhibitor, and pro‐ and anti‐fibrotic compounds on ECM synthesis, 3D collagen architecture, and mechanophenotype are quantified.

Multiphoton microfabrication and micropatternining (MMM)-based screening of multiplex cell niche factors for phenotype maintenance - Bovine nucleus pulposus cell as an example

Upon monolayer cultures on flat and rigid plastic dishes, many cells de-differentiate and lose their native phenotype. Technologies able to identify and reconstitute the cell niche factors that best maintain the physiological cellular phenotype in cultures are critical. We have developed a multiphoton microfabrication and micropatterning (MMM) technology, a robust 3D micro-printing platform capable to fabricate protein microstructures and micropatterns with quantitative, spatial and independent control of the mechanical, topological and extracellular matrix properties. Here, using bovine nucleus pulposus cells (bNPCs) as an example, we aim to reconstitute a spectrum of individual cell niche factors (2 mechanical, 9 topological and 4 matrices) in vitro for multiplex cell niche factor screening, and fabricate the optimal combinations of a series of shortlisted cell niche factors that best maintain the bNPC phenotype. Among all factors screened, two topological (micropillar array; fiber-bead structure) and two matrix (laminin; vitronectin) factors were shortlisted and the combinatory cell niche factors reconstituted from the shortlisted factors were found to synergistically augmented the expression of selected bNPC phenotype markers (Col II, SNAP25 and Keratin 8) and maintained their morphology and phenotype. These optimal cell niches can be micro-printed on culture dishes for physiologically relevant cultures and contribute to biomimetic scaffold design for intervertebral disc tissue engineering.

In Vitro Culture Expansion and Characterization of Buccal Mucosal Epithelial Cells for Tissue Engineering Applications in Urethral Stricture After Transportation Using a Thermoreversible Gelation Polymer.

Introduction: The transportation of tissues from hospitals to clinical laboratories for cell therapy is an essential component of regenerative medicine. Previously, we used laboratory-cultured mucosal cells from buccal epithelium expanded and encapsulated using a scaffold-hybrid approach to the urethral stricture (BEES-HAUS) procedure. In this study, to improve the outcomes, we compared the thermoreversible gelation polymer (TGP) transportation procedure with conventional culture methods, and reported its advantages. Methods: Human buccal mucosal tissues in Phase I of the study were transported in Euro-Collins solution (ECS) and the cells obtained were cultured in two-dimensional (2D) Dulbecco's modified Eagle's medium (DMEM), CnT-Prime epithelial 2D differentiation medium (CnT-PR), and a three-dimensional (3D)-TGP scaffold. In Phase II, tissues were transported in a TGP cocktail and the ECS. The cells were cultured in 2D-DMEM and 3D-TGP, quantified, and characterized by immunohistochemistry. Results: The cells in 3D-TGP culture maintained epithelial morphology in a better manner compared with 2D-DMEM, in which they developed fibroblast-like morphology. The TGP-transported cells grew rapidly. Immunohistochemical analysis results for AE1/AE3, EGFR, integrin-β1, p63, and p75 were intensely positive in 3D-TGP. Conclusion: The TGP-based cocktail used in human buccal tissue transportation yielded cells with better morphology maintenance. The TGP scaffold provides an optimal in vitro environment wherein epithelial cells better maintain their native phenotype compared to those cultured through conventional methods. These results suggest using TGP for the transportation and culture of human buccal tissues for clinical applications. In addition, the use of a TGP-based cocktail for the transport of other tissues for regenerative medicine applications is worth further analysis.

A Pulmonary Vascular Model From Endothelialized Whole Organ Scaffolds.

The development of an in vitro system for the study of lung vascular disease is critical to understanding human pathologies. Conventional culture systems fail to fully recapitulate native microenvironmental conditions and are typically limited in their ability to represent human pathophysiology for the study of disease and drug mechanisms. Whole organ decellularization provides a means to developing a construct that recapitulates structural, mechanical, and biological features of a complete vascular structure. Here, we developed a culture protocol to improve endothelial cell coverage in whole lung scaffolds and used single-cell RNA-sequencing analysis to explore the impact of decellularized whole lung scaffolds on endothelial phenotypes and functions in a biomimetic bioreactor system. Intriguingly, we found that the phenotype and functional signals of primary pulmonary microvascular revert back—at least partially—toward native lung endothelium. Additionally, human induced pluripotent stem cell-derived endothelium cultured in decellularized lung systems start to gain various native human endothelial phenotypes. Vascular barrier function was partially restored, while small capillaries remained patent in endothelial cell-repopulated lungs. To evaluate the ability of the engineered endothelium to modulate permeability in response to exogenous stimuli, lipopolysaccharide (LPS) was introduced into repopulated lungs to simulate acute lung injury. After LPS treatment, proinflammatory signals were significantly increased and the vascular barrier was impaired. Taken together, these results demonstrate a novel platform that recapitulates some pulmonary microvascular functions and phenotypes at a whole organ level. This development may help pave the way for using the whole organ engineering approach to model vascular diseases.

Bm-miR172c-5p Regulates Lignin Biosynthesis and Secondary Xylem Thickness by Altering the Ferulate 5 HydroxylaseGene in Bacopa monnieri.

MicroRNAs (miRNAs) are small non-coding, endogenous RNAs containing 20-24 nucleotides that regulate the expression of target genes involved in various plant processes. A total of 1,429 conserved miRNAs belonging to 95 conserved miRNA families and 12 novel miRNAs were identified from Bacopa monnieri using small RNA sequencing. The Bm-miRNA target transcripts related to the secondary metabolism were further selected for validation. The Bm-miRNA expression in shoot and root tissues was negatively correlated with their target transcripts. The Bm-miRNA cleavage sites were mapped within the coding or untranslated region as depicted by the modified RLM-RACE. In the present study, we validate three miRNA targets, including asparagine synthetase, cycloartenol synthase and ferulate 5 hydroxylase (F5H) and elucidate the regulatory role of Bm-miR172c-5p, which cleaves the F5H gene involved in the lignin biosynthesis. Overexpression (OE) of Bm-miR172c-5p precursor in B. monnieri suppresses F5H gene, leading to reduced lignification and secondary xylem thickness under control and drought stress. By contrast, OE of endogenous target mimics (eTMs) showed enhanced lignification and secondary xylem thickness leading to better physiological response under drought stress. Taken together, we suggest that Bm-miRNA172c-5p might be a key player in maintaining the native phenotype of B. monnieri under control and different environmental conditions.

A novel ultralow conductivity electromanipulation buffer improves cell viability and enhances dielectrophoretic consistency.

Cell separation has become a critical diagnostic, research, and treatment tool for personalized medicine. Despite significant advances in cell separation, most widely used applications require the use of multiple, expensive antibodies to known markers in order to identify subpopulations of cells for separation. Dielectrophoresis (DEP) provides a biophysical separation technique that can target cell subpopulations based on phenotype without labels and return native cells for downstream analysis. One challenge in employing any DEP device is the sample being separated must be transferred into an ultralow conductivity medium, which can be detrimental in retaining cells' native phenotypes for separation. Here, we measured properties of traditional DEP reagents and determined that after just 1-2h of exposure and subsequent culture, cells' viability was significantly reduced below 50%. We developed and tested a novel buffer (Cyto Buffer) that achieved 6 weeks of stable shelf-life and demonstrated significantly improved viability and physiological properties. We then determined the impact of Cyto Buffer on cells' dielectric properties and morphology and found that cells retained properties more similar to that of their native media. Finally, we vetted Cyto Buffer's usability on a cell separation platform (Cyto R1) to determine combined efficacy for cell separations. Here, more than 80% of cells from different cell lines were recovered and were determined to be >70% viable following exposure to Cyto Buffer, flow stimulation, electromanipulation, and downstream collection and growth. The developed buffer demonstrated improved opportunities for electrical cell manipulation, enrichment, and recovery for next generation cell separations.

A novel human donor cornea preservation cocktail incorporating a thermo-reversible gelation polymer (TGP), enhancing the corneal endothelial cell density maintenance and explant culture of corneal limbal cells

PurposeMcCarey-Kaufman’s (MK) medium and Optisol-GS medium are the most commonly employed media for human donor corneal preservation. In this study, we evaluated the preservation efficacy of discarded human donor corneas using a Thermo-reversible gelation polymer (TGP) added to these two media.MethodsThirteen human corneal buttons collected from deceased donors, which were otherwise discarded due to low endothelial cell density (ECD) were used. They were stored in four groups: MK medium, MK medium with TGP, Optisol-GS and Optisol-GS with TGP at 4 °C for 96 h. Slit lamp examination and specular microscopy were performed. Corneal limbal tissues from these corneas were then cultured using explant methodology one with and the other without TGP scaffold, for 21 days.ResultsMK + TGP and Optisol-GS + TGP preserved corneas better than without TGP, which was observed by maintenance of ECD which was significantly higher in Optisol-GS + TGP than MK + TGP (p-value = 0.000478) and corneal thickness remaining the same for 96 h. Viable corneal epithelial cells could be grown from the corneas stored only in MK + TGP and Optisol-GS + TGP. During culture, the TGP scaffold helped maintain the native epithelial phenotype and progenitor/stem cell growth was confirmed by RT-PCR characterization.ConclusionTGP reconstituted with MK and Optisol—GS media yields better preservation of human corneal buttons in terms of relatively higher ECD maintenance and better in vitro culture outcome of corneal limbal tissue. This method has the potential to become a standard donor corneal transportation-preservation methodology and it can also be extended to other tissue or organ transportation upon further validation.

Preliminary Study on the Development of In Vitro Human Respiratory Epithelium Using Collagen Type I Scaffold as a Potential Model for Future Tracheal Tissue Engineering

Pathological conditions of the tracheal epithelium, such as postoperative injuries and chronic conditions, often compromise the functionality of the respiratory epithelium. Although replacement of the respiratory epithelium using various types of tracheal transplantation has been attempted, there is no predictable and dependable replacement method that holds for safe and practicable long-term use. Therefore, we used a tissue engineering approach for ex vivo regeneration of the respiratory epithelium (RE) construct. Collagen type I was isolated from sheep tendon and it was fabricated in a three-dimensional (3D) scaffold format. Isolated human respiratory epithelial cells (RECs) and fibroblasts from nasal turbinate were co-cultured on the 3D scaffold for 48 h, and epithelium maturation was allowed for another 14 days in an air–liquid interface culture system. The scanning electron microscope results revealed a fabricated porous-structure 3D collagen scaffold. The scaffold was found to be biocompatible with RECs and fibroblasts and allows cells attachment, proliferation, and migration. Immunohistochemical analysis showed that the seeded RECs and fibroblasts were positive for expression of cytokeratin 14 and collagen type I markers, respectively, indicating that the scaffold supports the native phenotype of seeded cells over a period of 14 days. Although a longer maturation period is needed for ciliogenesis to occur in RECs, the findings suggest that the tissue-engineered RE construct is a potential candidate for direct use in tracheal epithelium replacement or tracheal tube reengineering.

Best Practices for Validation of Measurable Residual Disease Assessments By Multiparameter Flow Cytometry in Emerging Clinical Trials of Acute Myeloid Leukemia

Background: Measurable Residual Disease (MRD) assessments are gaining increasing acceptance as a prognostic factor for tailoring treatment in hematological malignancies. Acute Myeloid Leukemia (AML) is a heterogeneous disease with high relapse rates and presents a high unmet need for effective treatment options. Measurement of residual disease after therapy reflects a combination of all resistance mechanisms and is currently used for guiding treatment options. Study Design: In this study, we aimed to validate an AML-MRD assay by multiparameter flow cytometry (MFC) methodology. This is a 4-tube, 8-parameter assay designed to incorporate cell differentiation (CD) markers for identification of a diverse group (covering roughly 90% of patients, Cloos et al, 2018) of Leukemia Associated Immunophenotypes (LAIPs) to accurately identify both native phenotypes and phenotype shifts after drug treatment. These CD markers were selected based on extensive investigation of many markers and in line with the consensus recommendations from European Leukemia Network AML working party (Schuurhuis et al, 2018), while specimen testing and interpretation principles were performed in accordance with Cloos et al, 2018. The assay validation focused on evaluation of sensitivity (MRD cut point and LOD), precision and accuracy as key criteria for evaluating assay performance utilizing primary patient specimens and AML cell lines representing different LAIPs. The results were orthogonally verified in a blinded manner by morphologic assessment at Navigate and by the MRD-team at VUMC Amsterdam. Results: Two experimental approaches were adopted to evaluate analytical and functional sensitivity (clinical applicability) of the assay. Results indicated analytical sensitivity (LOD) as low as 0.01% LAIPs of total WBC and functional sensitivity (LOQ) of 0.1% (MRD cut point). Excellent repeatability and reproducibility (less than 20% CV) was observed across instruments, operators and independent measurements (n = 75). The frequencies of AML blasts detected by MFC and morphological examination were highly concordant (Spearman r = 0.95, P value < 0.001, n = 24). LAIPs deduced across nine patient specimens by the Navigate laboratory were independently confirmed by the MRD-team at VUMC Amsterdam. Conclusion: In summary, based on the use of consensus markers recommended by ELN for reliable capture of a broad group of LAIPs in AML patients and verification of key assay performance characteristics, we believe this comprehensive MFC based AML MRD assay is fit-for-purpose for accurately assessing measurable residual disease. Following clinical trial validation, MRD might be used as a surrogate endpoint for approval of emerging agents. Disclosures Marques Ramos: Novartis: Current Employment. Larson:BMS, Bioline, Celgene, Juno, Janssen: Research Funding; TORL Biotherapeutics: Current equity holder in private company. Sarikonda:Novartis: Current Employment.

Effective inhibition of Th17/Th22 pathway in 2D and 3D human models of psoriasis by Celastrol enriched plant cell culture extract

Psoriasis is an immune-mediated inflammatory disease in which the Th17 pathway is mainly involved. Systemic interventions with biologics that specifically block the Th17 pathway are effective to treat severe psoriasis. However, for efficient topical treatment, small molecules are more suitable than antibodies to penetrate and target epidermal keratinocytes, the key players in psoriasis. Celastrol, a well-described triterpene, is present in low amounts in Tripterygium wilfordii roots. By using plant cell culture (PCC), we were able to boost Celastrol production in bioreactors. Here, we evaluated immune modulator effect of Celastrol enriched extract (CEE) in Th17/Th22 psoriasis induced in 2D and 3D human models in vitro in view of its dermatological usage. Human CD4+ T cells (hCD4), Normal Human Epidermal Keratinocytes (NHEK), micro-epidermis and reconstructed human epidermis (RHE) were preincubated with CEE and reference controls. Then, hCD4 were stimulated by anti-[CD3/CD28] while others were stimulated by Th17/22 cytokines cocktails. Psoriasis biomarkers were assessed by ELISA (hCD4 and RHE), by RT-qPCR (NHEK) or by ICH/ELISA (micro-epidermis). In 2D stimulated models (hCD4 and NHEK), CEE dose dependently inhibited, respectively, the expression of Th17 cytokines and psoriasis induced biomarkers. In 3D models (RHE and micro-epidermis), IL-8 expression was significantly reduced (RHE) and native phenotype was restored by CEE (micro-epidermis). These results clearly showed that Th17/Th22 cytokines, main inflammatory parameters, and psoriasis associated key biomarkers were inhibited by CEE in both 2D and 3D human in vitro models. Therefore, skin homeostasis could be restored by these modulator effects. Moreover, this high added value CEE was obtained by an ecofriendly bioprocess in contrast to traditional roots extracts. This is the first time that a well-defined CEE immune modulator has been proposed for psoriasis adjuvant care to reduce inflammation.

236 Celastrol enriched extract modulates Th17 key-disease mediators by interrupting feed-forward inflammation and by restoring homeostasis in psoriasis epidermal skin

Background: Psoriasis is an autoinflammatory skin disorder due to complex interaction between lymphocytes, DCs and keratinocytes (KCs). High IL-17 level produced by T cells, induces a feed-forward inflammatory response in KCs, resulting in the development of thickened skin lesions infiltrated with mixture of inflammatory cell population. Here, we evaluated modulator effect of Celastrol enriched extract (CEE), from T. wilfordii plant cell culture, in two Th17 psoriasis induced models in vitro. Methods: NHEK were stimulated by cytokines cocktail Mix 1 [IL-17, OSM and TNF-α] for 24 H after a preincubation for 4 H with: CEE (90 ng/ml), Celastrol (135 ng/ml) or JAK I inhibitor (10 μM). Anti-microbial peptides (AMPs), Cytokines and Chemokines key biomarkers gene were assessed by RT-qPCR. Multilayered micro-epidermis model (Cytoo™) were stimulated by cytokines cocktail Mix 2 [Mix 1 + IL-22 & IL-1α] for 3 days after preincubation for 24 H with: CEE (10 ng/ml) or Tazarotene (0.3 μM). The readouts assayed were IL-1β (ELISA) and Filaggrin (Flg) or S100A7 (immunostainings). CEE is expressed as Celastrol titer in all assays. Results and conclusion: In NHEK, CEE and Celastrol inhibited expression of biomarkers: AMPs (S100A7, PI3, DEFB4), Cytokines (IL-19 & -36 g) and Chemokines (CXCL-1 & -8, CCL-5 & -20). These biomarkers, up-regulated and self-induced by KCs, are described as key mediators in maintaining feed-forward inflammation in psoriasis. In micro-epidermis, CEE inhibited significantly IL-1β and S100A7 while up-regulated Flg expression. CEE, a high added-value product has effective modulator effects in Th17 mediated epidermal skin. It breaks the vicious psoriasis inflammation circle and restores epidermal native phenotype. This is the first time, a well-defined immune-modulator CEE natural of origin, has been proposed for dermo cosmetic in psoriasis.

Analysis of the performance of the ultra-diluted Viscum album in cultivation of mesenchymal stem cells and mammary adenocarcinoma cells pmc-42 and mcf-7

Adenocarcinoma in breast can be of several types and PMC-42 and MCF-7 cell lines are a well-established cell lines used in in vitro studies retaining much of the breast's native phenotype. Viscum album, a plant which extract has cytotoxic effects, is included in the brand-new class of cancer therapies and its ultra-diluted version is a promising area of interest. The MTT assay was realized to evaluate the cytotoxic potential and calculate the inhibitory concentration of different concentrations of ultra-diluted Viscum album D30 (VAD30). The results indicated that 60mL/mL, 37mL/mL and 35ml/mL of VAD30 were cytotoxic to 50% of mesenchymal stem cell, MCF-7 and PMC-42 cells respectively. This result shows the better potency of action of VAD30 against tumor cells, as a lower concentration inhibits the growing of this cell line in comparison to health cells, demonstrating that this medicine has the potential to be used in cancer therapy.