Development and Optimization of a Lactate Dehydrogenase Assay Adapted to 3D Cell Cultures

In recent years, 3D cell culture systems have emerged as sophisticated in vitro models, providing valuable insights into human physiology and diseases. The transition from traditional 2D to advanced 3D cultures has introduced novel obstacles, complicating the characterization and analysis of these models. While the lactate dehydrogenase (LDH) activity assay has long been a standard readout for viability and cytotoxicity assessments in 2D cultures, its applicability in long-term 3D cultures is hindered by inappropriate normalization and low LDH stability over time. In response to these challenges, we propose an optimization of LDH assays, including a crucial normalization step based on total protein quantification and a storage method using an LDH preservation buffer. We applied it to compare unexposed cerebral organoids with organoids exposed to a toxic dose of valproic acid, and showed efficient normalization of cellular viability as well as enhanced LDH stability within the buffer. Importantly, normalized LDH activity results obtained were independent of organoid dimension and cell density. This refined LDH assay, tailored to address 3D culture constraints, allows for the transposition of this routine test from 2D to 3D cultures

A new immunosensor for breast cancer cell detection using antibody-coated long alkylsilane self-assembled monolayers in a parallel plate flow chamber.

International audienceWe designed a new efficient and reliable immunosensor and demonstrated its analytic performance to capture breast cancer MCF7 and T47D cells, under laminar flow, onto antibody-coated long alkylsilane self-assembled monolayers (SAMs) in a parallel plate flow chamber. The surface floor of the laminar flow chamber was grafted with an amino-terminated long alkyl chain spacer, 21-aminohenicosyl trichlorosilane (AHTS) followed by tethering a specific monoclonal antibody directed against the human epithelial cell adhesion molecule (EpCAM) antigen, which is overexpressed in primary breast cancer. Properties of the AHTS- and antibody-grafted surface floor were compared to that of surface floors coated with the short alkyl spacers 3-glycidoxy-propyl trimethoxysilane (GPTS) or 3-aminopropyl triethoxysilane (APTES) and antibodies. A theoretical model was constructed according to the geometry of the flow chamber in order to calculate the trajectories that would use cell flows. Cell capture experiments demonstrated that cell immobilization was optimized throughout the whole flow chamber. High cell capture was yielded on antibody-tethered long alkyl AHTS surface. This new procedure offers multiple advantages: a versatile tool readily applied to a panel of purified antibodies, an enrichment of cell immobilization using repetitive cell flow, and a stable capturing surface suitable for long term storage and handling

Histological characterization of liver involvement in systemic mastocytosis.

International audienceBackground and AimsSystemic mastocytosis (SM) is characterized by the accumulation of atypical mast cells (MCs) in organs. Liver histology of SM has been marginally described and accurate histological classification is critical, given the consequences of aggressive SM diagnosis. We aimed to describe the histological features associated with liver SM using updated tools.MethodsUsing the database of the French Reference Centre for Mastocytosis, we retrospectively identified patients with a liver biopsy (LB) and a diagnosis of SM. All LB procedures were performed according to the local physician in charge and centrally reviewed by an expert pathologist.ResultsA total of 28 patients were included: 6 had indolent SM, 9 had aggressive SM, and 13 had SM with an associated hematologic neoplasm. Twenty-five (89%) patients presented hepatomegaly, and 19 (68%) had portal hypertension. The LB frequently showed slight sinusoid dilatation (82%). Fibrosis was observed in 3/6 indolent SM and in almost all advanced SM cases (21/22), but none of them showed cirrhosis. A high MC burden (>50 MCs/high-power field) was correlated with elevated blood alkaline phosphatase levels (p = .030). The presence of portal hypertension was associated with a higher mean fibrosis grade (1.6 vs. 0.8 in its absence; p = .026). In advanced SM, the presence of nodular regenerative hyperplasia (NRH) was associated with decreased overall survival (9.5 vs. 46.3 months, p = .002).ConclusionsMC infiltration induced polymorphic hepatic lesions and the degree of fibrosis is associated with portal hypertension. NRH identifies a poor prognosis subgroup of patients with advanced SM. Assessing liver histology can aid in SM prognostic evaluation