Generating Bovine Monocyte-Derived Dendritic Cells for Experimental and Clinical Applications Using Commercially Available Serum-Free Medium

Abstract

Advances in fundamental and applied immunology research often originate from pilot studies utilizing animal models. While cattle represent an ideal model for disease pathogenesis and vaccinology research for a number of human disease, optimized bovine culture models have yet to be fully established. Monocyte-derived dendritic cells (MoDC) are critical in activating adaptive immunity and are an attractive subset for experimental and clinical applications. The use of serum-supplemented culture medium in this ex vivo approach is undesirable as serum contains unknown quantities of immunemodulating components and may induce unwanted immune responses if not autologous. Here, we describe a standardized protocol for generating bovine MoDC in serum-free medium (AIM-V) and detail the MoDC phenotype, cytokine profile, and metabolic signature achieved using this culture methodology. MoDC generated from adult, barren cattle were used for a series of experiments that evaluated the following culture conditions: medium type, method of monocyte enrichment, culture duration, and concentration of differentiation additives. Viability and yield were assessed using flow cytometric propidium iodide staining and manual hemocytometer counting, respectively. MoDC phenotype and T cell activation and proliferation were assessed by flow cytometric analysis of surface markers (MHC class II, CD86, CD14, and CD205), and CD25 and CFSE respectively. Cytokine secretion was quantified using a multiplex bovine cytokine panel (IL-1a, IL-1b, IL-8, IL-10, IL-17A, IFN-g, MIP-1a, TNF-a, and IL-4). Changes in cell metabolism following stimulation were analyzed using an Extracellular Flux (XFe96) Seahorse Analyzer. Data were analyzed using paired t-tests and repeated measures ANOVA. Immature MoDC generated in serum-free medium using magnetic-activated cell sorting with plate adhesion to enrich monocytes and cultured for 4 days have the following phenotypic profile: MHC class II +++ , CD86 + , CD205 ++ , and CD14-. These MoDC can be matured with PMA and ionomycin as noted by increased CD86 and CD40 expression, increased cytokine secretion (IL-1a, IL-10, MIP-1a, and IL-17A), a metabolic switch to aerobic glycolysis, and induction of T cell activation and proliferation following maturation. Cultivation of

Simplified Approaches for the Production of Monocyte-Derived Dendritic Cells and Study of Antigen Presentation in Bovine

“…In serum-free conditions, moDCs showed CD11c and CD172a expression and antigen presentation capacity comparable to counterparts produced in FBS-supplemented medium. Nevertheless, contrary to a previous report ( 28 ), bovine moDCs produced in AIM V presented lower MHC-II expression than in RPMI-FBS medium.…”

Review of Dendritic Cells, Their Role in Clinical Immunology, and Distribution in Various Animal Species

“…This method was originally described in several human studies for DC generation; however, it has been adapted for use in cattle. Recently, monocyte-derived bovine DCs have been generated from experimental and clinical applications using serum-free media [ 75 ]. moDCs provide a further valuable model that does not involve the use of prolonged isolation procedures.…”

Modern Methods of Obtaining Immune Dendritic Cells With Anti-Tumor Potential

“…Що стосується самої сироватки, то її додавання (якщо вона не аутологічна) в культуральне середовище при індукуванні імунних ДК може спричинити небажані ефекти, оскільки сироватка може містити поки що невідомі імуномодулювальні компоненти. У зв'язку з цим деякі дослідники для отримання імунних ДК із моноцитів використовували культуральне середовище без сироватки [98,99]. Як було продемонстровано J. Calmeiro та співавт.…”