Improving assay feasibility and biocompatibility of 3D cyclic olefin copolymer microwells by superhydrophilic modification via ultrasonic spray deposition of polyvinyl alcohol.

Sample partitioning is a crucial step towards digitization of biological assays on polymer microfluidic platforms. However, effective liquid filling into microwells and long-term hydrophilicity remain a challenge in polymeric microfluidic devices, impeding the applicability in diagnostic and cell culture studies. To overcome this, a method to produce permanent superhydrophilic 3-dimensional microwells using cyclic olefin copolymer (COC) microfluidic chips is presented. The COC substrate is oxidized using UV treatment followed by ultrasonic spray coating of polyvinyl alcohol solution, offering uniform and long-term coating of high-aspect ratio microfeatures. The coated COC surfaces are UV-cured before bonding with a hydrophobic pressure-sensitive adhesive to drive selective filling into the wells. The surface hydrophilicity achieved using this method remains unchanged (water contact angle of 9°) for up to 6 months and the modified surface is characterized for physical (contact angle & surface energy, morphology, integrity of microfeatures and roughness), chemical composition (FTIR, Raman spectroscopy) and coating stability (pH, temperature, time). To establish the feasibility of the modified surface in biological applications, PVA-coated COC microfluidic chips are tested for DNA sensing (digital LAMP detection of CMV), and biocompatibility through protein adsorption and cell culture studies (cell adhesion, viability, and metabolic activity). Kidney and breast cells remained viable for the duration of testing (7 days) on this modified surface, and the coating did not affect the protein content, morphology or quality of the cultured cells. The ultrasonic spray coated system, coating with 0.25 % PVA for 15 cycles with 0.12 A current after UV oxidation, increased the surface energy of the COC (naturally hydrophobic) from 22.04 to 112.89 mJ/m2 and improved the filling efficiency from 40 % (native untreated COC) to 94 % in the microwells without interfering with the biocompatibility of the surface, proving to be an efficient, high-throughput and scalable method of microfluidic surface treatment for diagnostic and cell growth applications.

Fungal cryopreservation across 61 genera: Practical application and method evaluation.

Fungi occupy important environmental, cultural, and socioeconomic roles. However, biological research of this diverse kingdom has lagged behind that of other phylogenetic groups. This is partially the result of the notorious difficulty in culturing a diverse array of filamentous fungal species due to their (i) often unpredictable growth, (ii) unknown preferences for culturing conditions, and (iii) long incubation times compared with other microorganisms such as bacteria and yeasts. Given the complexity associated with concurrently culturing diverse fungal species, developing practical methods for preserving as many species as possible for future research is vital. The widely accepted best practice for preserving fungal tissue is the use of cryogenic biobanking at -165 C, allowing for the preservation and documentation of stable genetic lineages, thus enabling long-term diversity-centered research. Despite the extensive literature on fungal cryopreservation, substantial barriers remain for implementation of cryogenic biobanks in smaller mycological laboratories. In this work, we present practical considerations for the establishment of a fungal culture biobank, as well as provide evidence for the viability of 61 fungal genera in cryogenic storage. By providing a pragmatic methodology for cryogenically preserving and managing many filamentous fungi, we show that creating a biobank can be economical for independently owned and operated mycology laboratories, which can serve as a long-term resource for biodiversity, conservation, and strain maintenance.

Factors of presenteeism and its association with detrimental effects among employees in Switzerland working in different sectors - a cross-sectional study using a multi-item instrument.

PURPOSE: Presenteeism, the phenomenon of employees working despite illness, is a significant issue globally, impacting individual well-being and organizational efficiency. This study examines presenteeism among Swiss employees, exploring its occurrence, primary factors, reasons, and impact on employees' health. METHODS: This study used cross-sectional data from 1,521 employees in different sectors in Switzerland. Descriptive statistics and multiple linear models for influencing factors and detrimental effects, such as burnout symptoms, job satisfaction, general health, and quality of life, were calculated for data analysis. Presenteeism was measured using the Hägerbäumer multi-item scale, ranging from 1 = "Never in case of illness" - 5 = "Very often in case of illness." RESULTS: The employees reported that in case of illness, they rarely worked in the last 12 months M = 2.04 (SD = 1.00). A positive approach to presenteeism in the team was associated with less presenteeism (ß = -0.07) and problematic leadership culture in dealing with presenteeism with increased presenteeism (ß = 0.10). In addition to well-known factors, presenteeism was significant for burnout symptoms (ß = 1.49), general health status (ß = -1.5), and quality of life (ß = -0.01). CONCLUSION: The study offers insights into the phenomenon of presenteeism among Swiss employees in various sectors by applying a multi-item scale for presenteeism. The findings indicate that a positive team dynamic and organizational culture may significantly reduce presenteeism. Presenteeism behavior is a significant factor of adverse outcomes. This highlights the importance of acknowledging presenteeism in the context of occupational health.

Adaptive Laboratory Evolution to Improve Recombinant Protein Production Using Insect Cells.

Adaptive laboratory evolution (ALE) is a powerful tool for enhancing the fitness of cell lines in specific applications, including recombinant protein production. Through adaptation to nonstandard culture conditions, cells can develop specific traits that make them high producers. Despite being widely used for microorganisms and, to lesser extent, for mammalian cells, ALE has been poorly leveraged for insect cells. Here, we describe a method for adapting insect High Five and Sf9 cells to nonstandard culture conditions via an ALE approach. Aiming to demonstrate the potential of ALE to improve productivity of insect cells, two case studies are demonstrated. In the first, we adapted insect High Five cells from their standard pH (6.2) to neutral pH (7.0); this adaptation allowed to improve production of influenza virus-like particles (VLPs) by threefold, using the transient baculovirus expression vector system. In the second, we adapted insect Sf9 cells from their standard culture temperature (27 °C) to hypothermic growth (22 °C); this adaptation allowed to improve production of influenza VLPs by sixfold, using stable cell lines. These examples demonstrate the potential of ALE for enhancing productivity within distinct insect cell hosts and expression systems by manipulating different culture conditions.

Recombinant AAV Production.

The insect cell-baculovirus expression vector (IC-BEV) platform has enabled small research-scale and large commercial-scale production of recombinant proteins and therapeutic biologics including recombinant adeno-associated virus (rAAV)-based gene delivery vectors. The wide use of this platform is comparable with other mammalian cell line-based platforms due to its simplicity, high-yield, comparable quality attributes, and robust bioprocessing features. In this chapter, we describe a rAAV production protocol employing one of the recent modifications of the One-Bac platform that consists of a stable transformed Sf9 cell line carrying AAV Rep2/Cap5 genes that are induced upon infection with a single recombinant baculovirus expression vector harboring the transgene of interest (rAAV genome). The overall protocol consists of essential steps including rBEV working stock preparation, rAAV production, and centrifugation-based clarification of cell culture lysate. The same protocol can also be applied for rAAV vector production using traditional Three-Bac, Two-Bac, and Mono-Bac platforms without requiring significant changes.

Protein Production at the 100L Scale.

The Baculovirus Expression Vector System (BEVS) has revolutionized the field of recombinant protein expression by enabling efficient and high yield production. The platform offers many advantages including manufacturing speed, flexible design, and scalability. In this chapter, we describe the methods including strategies and considerations to successfully optimize and scale-up using BEVS as a tool for production (Fig. 1). As an illustrative case study, we present an example focused on the production of a viral glycoprotein.

Recombinant AAV Purification.

Purification of rAAV is a crucial unit operation of the AAV production process. It enables the capture of AAV and removal of contaminants such as host cell proteins, host cell DNA, and other cell culture-related impurities. Here we describe the purification of rAAV produced in insect cells Sf9/rBEV by immuno-affinity capture chromatography. The method is fully scale-amenable unlike other traditional purification methods based on ultracentrifugation. The method reported herein has two main steps: (1) the clarification of cell lysate by depth filtration and (2) the selective capture and single-step purification of AAV via immune-affinity chromatography. This purification method has been successfully implemented to purify the majority of wild-type AAV serotypes.

TCID50 Assay: A Simple Method to Determine Baculovirus Titer.

There are many methods that can be used to determine the infectious titer of your baculovirus stock. The TCID50 method is a simple end-point dilution method that determines the amount of baculovirus virus needed to produce a cytopathic effect or kill 50% of inoculated insect cells. Serial dilutions of baculovirus stock are added to Sf9 cells cultivated in 96-well plates and 3-5 days after infection, cells are monitored for cell death or cytopathic effect. The titer can then be calculated by the Reed-Muench method as described in this method.

Functional Baculovirus Particle Quantification via Plaque Assay.

Plaque assay method enables the quantification of infectious baculovirus when defined as plaque forming units (PFU). It allows to determine the amount of infectious virus needed to infect the cells at a specific multiplicity of infection (MOI). Serial dilutions of baculovirus stock are added to the Sf9 cells monolayer followed by addition of 5% Agarose overlay. Six days after infection clear infection halos are observed using a neutral red solution. Here we describe the quantification of recombinant baculovirus expression vector (rBEV) carrying a transgene in an rAAV expression cassette. Reproducible quantification of PFU is obtained with this method.

Fungal endo and exochitinase production, characterization, and application for Candida biofilm removal.

Chitinases are promising enzymes for a multitude of applications, including chitooligosaccharide (COS) synthesis for food and pharmaceutical uses and marine waste management. Owing to fungal diversity, fungal chitinases may offer alternatives for chitin degradation and industrial applications. The rapid reproduction cycle, inexpensive growth media, and ease of handling of fungi may also contribute to reducing enzyme production costs. Thus, this study aimed to identify fungal species with chitinolytic potential and optimize chitinase production by submerged culture and enzyme characterization using shrimp chitin. Three fungal species, Coriolopsis byrsina, Trichoderma reesei, and Trichoderma harzianum, were selected for chitinase production. The highest endochitinase production was achieved in C. byrsina after 168 h cultivation (0.3 U mL- 1). The optimal temperature for enzyme activity was similar for the three fungal species (up to 45 and 55 ºC for endochitinases and exochitinases, respectively). The effect of pH on activity indicated maximum hydrolysis in acidic pH (4-7). In addition, the crude T. reesei extract showed promising properties for removing Candida albicans biofilms. This study showed the possibility of using shrimp chitin to induce chitinase production and enzymes that can be applied in different industrial sectors.

Up you get: Norwegian parents' reactions to children's negative emotions.

INTRODUCTION: Developmental research suggests that children learn to regulate their emotions and behavior through a process of emotion socialization. The main body of literature is based on samples from the United States, and very little is known about the socialization of emotions in Nordic settings. OBJECTIVES: The current study aimed to explore associations between mothers' and fathers' reactions to children's negative emotions and externalizing behavior problems in a Nordic cultural context, and to explore gender differences in these associations. METHODS: Parent-report data on the Coping with Children's Negative Emotions Scale (CCNES) and the Eyberg Child Behavior Inventory 6 (ECBI) was collected in a large sample of Norwegian preschool-aged children (mothers, n = 242; fathers, n = 183; N = 257; M = 54 months, SD = 4.54; 49% boys). Teacher-report data was collected using the Strengths and Difficulties Questionnaire (SDQ) conduct scale (n = 117). RESULTS: Both parents' supportive and non-supportive reactions were associated with child externalizing difficulties in expected directions as evidenced by path models, controlling for socioeconomic status and age. A pattern emerged in which non-supportive reactions to a greater extent predicted an increase in externalizing problems in girls, and supportive reactions predicted lower levels of externalizing problems in boys. CONCLUSION: Our findings supported the basic assumptions of emotion socialization theory in a Nordic cultural context in which parental supportive and non-supportive responses are related to child externalizing difficulties. Nordic parents are important socialization agents for their children, but their behaviors had a differential effect on boys' and girls' externalizing behavior problems.

Conductive extracellular matrix derived/chitosan methacrylate/ graphene oxide-pegylated hybrid hydrogel for cell expansion.

Electrical stimulation has emerged as a cornerstone technique in the rapidly evolving field of biomedical engineering, particularly within the realms of tissue engineering and regenerative medicine. It facilitates cell growth, proliferation, and differentiation, thereby advancing the development of accurate tissue models and enhancing drug-testing methodologies. Conductive hydrogels, which enable the conduction of microcurrents in 3D in vitro cultures, are central to this advancement. The integration of high-electroconductive nanomaterials, such as graphene oxide (GO), into hydrogels has revolutionized their mechanical and conductivity properties. Here, we introduce a novel electrostimulation assay utilizing a hybrid hydrogel composed of methacryloyl-modified small intestine submucosa (SIS) dECM (SISMA), chitosan methacrylate (ChiMA), and GO-polyethylene glycol (GO-PEG) in a 3D in vitro culture within a hypoxic environment of umbilical cord blood cells (UCBCs). Results not only demonstrate significant cell proliferation within 3D constructs exposed to microcurrents and early growth factors but also highlight the hybrid hydrogel's physiochemical prowess through comprehensive rheological, morphological, and conductivity analyses. Further experiments will focus on identifying the regulatory pathways of cells subjected to electrical stimulation.

The Association of Transformational Leadership on Safety Practices Among Nurses: The Mediating Role of Patient Safety Culture.

Background: To ensure best possible patient outcomes, patient safety is a major component of healthcare delivery system that needs to be prioritized. Safety practices among nurses are essential to maintain patient safety, especially the practices of medication administration, handover, patient falls and unplanned extubations prevention. Purpose: To investigate the mediating effect of patient safety culture between the relationship of transformational leadership and safety practices among nurses. Methods: The data in this cross-sectional study were gathered from a survey targeted clinical nurses using a random sampling technique. The study was conducted in a medical city in Saudi Arabia, and two hundred nurses were surveyed. The Multifactor Leadership, Hospital Survey on Patient Safety Culture, and Nursing Safety Practice questionnaires were used in the study. Results: The results revealed significant positive associations between transformational leadership, patient safety culture, and nursing safety practices. Moreover, patient safety culture mediates the association between transformational leadership and safety practices among nurses. Conclusion: Enhancing transformational leadership capabilities among nurse managers should be considered in order to improve nursing safety practices. Additionally, patient safety culture should be measured and improved periodically to ensure better nursing safety practices.

Informal institutions and corporate innovation: A Perspective from Red Culture.

Since the 20th century, Red Culture has served as a significant informal institution guiding revolutionary trajectory and developmental course. However, integrating Red Culture into contemporary corporate management and leveraging its constructive influence within today's market-driven economy necessitates comprehensive exploration and thoughtful consideration. This study aims to explore the potential influence of Red Culture on contemporary innovation. Empirical findings reveal substantial and affirmative effects of Red Culture on corporate innovation. Specifically, a heightened Red Culture ambiance correlates with a marked increase in both innovation input and output within corporate. Further investigation underscores Red Culture's pivotal governance role in mitigating strategic manipulation of innovation and research and development practices, especially within the overarching framework of innovation-driven strategies. Moreover, Red Culture synergizes with formal innovation incentive mechanisms, jointly fostering corporate innovation. This study provides micro-level empirical evidence that elucidates the impact of Red Culture on corporate innovation. Additionally, it furnishes valuable policy insights for the practical implementation and enhancement of pertinent Red Culture initiative.

Establishment of a humanized mouse model using steady-state peripheral blood-derived hematopoietic stem and progenitor cells facilitates screening of cancer-targeted T-cell repertoires.

Background: Cancer-targeted T-cell receptor T (TCR-T) cells hold promise in treating cancers such as hematological malignancies and breast cancers. However, approaches to obtain cancer-reactive TCR-T cells have been unsuccessful. Methods: Here, we developed a novel strategy to screen for cancer-targeted TCR-T cells using a special humanized mouse model with person-specific immune fingerprints. Rare steady-state circulating hematopoietic stem and progenitor cells were expanded via three-dimensional culture of steady-state peripheral blood mononuclear cells, and then the expanded cells were applied to establish humanized mice. The human immune system was evaluated according to the kinetics of dendritic cells, monocytes, T-cell subsets, and cytokines. To fully stimulate the immune response and to obtain B-cell precursor NAML-6- and triple-negative breast cancer MDA-MB-231-targeted TCR-T cells, we used the inactivated cells above to treat humanized mice twice a day every 7 days. Then, human T cells were processed for TCR ß-chain (TRB) sequencing analysis. After the repertoires had been constructed, features such as the fraction, diversity, and immune signature were investigated. Results: The results demonstrated an increase in diversity and clonality of T cells after treatment. The preferential usage and features of TRBV, TRBJ, and the V-J combination were also changed. The stress also induced highly clonal expansion. Tumor burden and survival analysis demonstrated that stress induction could significantly inhibit the growth of subsequently transfused live tumor cells and prolong the survival of the humanized mice. Conclusions: We constructed a personalized humanized mouse model to screen cancer-targeted TCR-T pools. Our platform provides an effective source of cancer-targeted TCR-T cells and allows for the design of patient-specific engineered T cells. It therefore has the potential to greatly benefit cancer treatment.

U.S. primary care physician perceptions on barriers to providing guideline-driven care for UTI and recurrent UTI: a qualitative study.

BACKGROUND: Urinary tract infections (UTI) affect almost two-thirds of all women during their lives and many experience recurrent infections. There are evidence-based guidelines from multiple international societies for evaluation and treatment; however, recent claims-based analyses have demonstrated that adherence to these guidelines is poor. This study seeks to understand the barriers experienced by U.S. primary care providers (PCPs) to providing guideline-based care for UTI and recurrent UTI (rUTI). METHODS: Semi-structured interviews of 18 PCPs, recruited from the greater Los Angeles area, examined real-world clinical management of UTI/rUTI episodes, decisions to refer to subspecialty care, and resources guiding counseling and management. Grounded theory methodology served to analyze interview transcripts and identify preliminary and major themes. RESULTS: Participants expressed the desire to obtain urine cultures for each cystitis episode, but felt pressured to make compromises by patient demands or barriers to care. PCPs had lower thresholds to empirical treatment if patients had a history of rUTIs, were elderly, or declined evaluation. Laboratory data was minimally utilized in clinical decision-making: urinalyses were infrequently considered when interpreting culture data. PCPs treated a broad set of urologic and non-urologic symptoms as UTI, even with negative cultures. PCPs did not feel comfortable initiating UTI prophylaxis, instead seeking specialist evaluation for anatomic causes. They were unaware of management guidelines, typically utilizing UpToDate® as their primary resource. Few evidence-based UTI prevention interventions were recommended by providers. CONCLUSIONS: Low availability of succinct and clear professional guidelines are substantial barriers to appropriate UTI/rUTI care. Poor useability of clinical guidance documents results in substantial confusion about the role of preventative measures and additional diagnostic testing. Difficulties in patient access to care providers lead to expectations for presumptive treatment. Future studies are needed to determine if improved educational materials for providers and/or management algorithms can improve guideline concordance of UTI management.

Diagnostic Usefulness of Liquid Culture Medium for Mycobacterium avium-intracellulare Complex Lung Disease: A Single-Centre, Retrospective Study.

Background The diagnosis of Mycobacterium avium-intracellulare complex lung disease (MAC-LD) requires two or more positive sputum cultures. Few reports have examined the usefulness of adding liquid culture to conventional solid culture for diagnosing MAC-LD. Methods A retrospective, cohort study of patients examined at Kurashiki Central Hospital in Japan with a confirmed diagnosis of MAC-LD between January 1, 2002, and June 20, 2021, was conducted. The primary endpoint was the culture positivity rate, which was compared between the liquid and Ogawa culture media in patients who underwent sputum culture using both methods. Secondary endpoints were the culture positivity rate in smear-positive specimens and the positivity rate by radiological type. Results The study, which involved 351 patients and 702 specimens, showed a higher positivity rate for liquid culture (n=690, 98.3%) than Ogawa culture (n=315, 44.9%). Overall, 265 patients (75.5%) would have had delayed MAC-LD diagnosis without liquid medium being used. Of the 95 smear-positive specimens, 71 (74.7%) were positive on both cultures, whereas 24 (25.3%) were positive only on liquid culture. The positivity rate of Ogawa culture varied by radiological type. Conclusions Liquid culture is more valuable for the early diagnosis of MAC-LD than Ogawa culture.

Mirage de tuberculose in the 21st century.

The occurrence of transient culture positivity for Mycobacterium tuberculosis (MTB), known as mirage de tuberculose, poses significant challenges in understanding its spectrum and implications. Here, we report a case of transient culture positivity, oscillating between detectable and non-detectable MTB cultures with minimal radiological features and review the literature on this phenomenon. The scarcity of scientific literature on this subject stems from the inherent impossibility of systematically studying mirage de tuberculose. Ethical and public health concerns prevent withholding treatment to monitor spontaneous reversion to negative cultures. Based on the literature, we estimate that mirage de tuberculose occurs in approximately one-third of individuals infected with MTB who exhibit no symptoms. Despite the inherently limited nature of these findings, they suggest that the significance of mirage de tuberculose may be greater than currently perceived. Managing cases of mirage de tuberculose presents formidable challenges from a public health perspective. Striking a balance between prompt treatment initiation to prevent transmission and the risk of unnecessary treatment requires careful consideration. In conclusion, mirage de tuberculose remains a poorly understood clinical entity with very limited literature available. Advancing research and interdisciplinary collaborations are essential to unravel the intricacies of this phenomenon and develop effective strategies to address its public health challenges.

L'apparition d'une culture transitoire positive pour Mycobacterium tuberculosis (MTB), connue sous le nom de mirage de tuberculose, pose des défis importants dans la compréhension de son spectre et de ses implications. Nous rapportons ici un cas de positivité transitoire des cultures, oscillant entre des cultures MTB détectables et non détectables avec des caractéristiques radiologiques minimales et passons en revue la littérature sur ce phénomène. La rareté de la littérature scientifique sur ce sujet provient de l'impossibilité inhérente d'étudier systématiquement le mirage de tuberculose. Des préoccupations éthiques et de santé publique empêchent l'interruption du traitement pour surveiller le retour spontané à des cultures négatives. Sur la base de la littérature, nous estimons que le mirage de tuberculose survient chez environ un tiers des personnes infectées par le MTB qui ne présentent aucun symptôme. Malgré la nature intrinsèquement limitée de ces résultats, ils suggèrent que l'importance du mirage de tuberculose pourrait être plus grande que ce que l'on perçoit actuellement. La prise en charge des mirages de tuberculose présente des défis considérables du point de vue de la santé publique. Il faut trouver un équilibre entre l'instauration rapide du traitement pour prévenir la transmission et le risque d'un traitement inutile. En conclusion, le mirage de tuberculose reste une entité clinique mal comprise et la littérature disponible est très limitée. L'avancement de la recherche et les collaborations interdisciplinaires sont essentiels pour démêler les subtilités de ce phénomène et élaborer des stratégies efficaces pour relever ses défis en matière de santé publique.

Culture-Free Whole Genome Sequencing of Mycobacterium tuberculosis Using Ligand-Mediated Bead Enrichment Method.

Background: Direct whole genome sequencing (WGS) of Mycobacterium tuberculosis (Mtb) can be used as a tool to study drug resistance, mixed infections, and within-host diversity. However, WGS is challenging to obtain from clinical samples due to low number of bacilli against a high background. Methods: We prospectively collected 34 samples (sputum, n = 17; bronchoalveolar lavage, n = 13; and pus, n = 4) from patients with active tuberculosis (TB). Prior to DNA extraction, we used a ligand-mediated magnetic bead method to enrich Mtb from clinical samples and performed WGS on Illumina platform. Results: Mtb was definitively identified based on WGS from 88.2% (30/34) of the samples, of which 35.3% (12/34) were smear negative. The overall median genome coverage was 15.2% (interquartile range [IQR], 7.7%-28.2%). There was a positive correlation between load of bacilli on smears and genome coverage (P < .001). We detected 58 genes listed in the World Health Organization mutation catalogue in each positive sample (median coverage, 85% [IQR, 61%-94%]), enabling the identification of mutations missed by routine diagnostics. Mutations causing resistance to rifampicin, isoniazid, streptomycin, and ethambutol were detected in 5 of 34 (14.7%) samples, including the rpoB S441A mutation that confers resistance to rifampicin, which is not covered by Xpert MTB/RIF. Conclusions: We demonstrate the feasibility of magnetic bead-based enrichment for culture-free WGS of Mtb from clinical specimens, including smear-negative samples. This approach can also be integrated with low-cost sequencing workflows such as targeted sequencing for rapid detection of Mtb and drug resistance.

The systemic cellular immune response against allogeneic mesenchymal stem cells is influenced by inflammation, differentiation and MHC compatibility: in vivo study in the horse.

The effectiveness and safety of allogeneic mesenchymal stem/stromal cells (MSCs) can be affected by patient's immune recognition. Thus, MSC immunogenicity and their immunomodulatory properties are crucial aspects for therapy. Immune responses after allogeneic MSC administration have been reported in different species, including equine. Interactions of allogenic MSCs with the recipient's immune system can be influenced by factors like matching or mismatching for the major histocompatibility complex (MHC) between donor-recipient, and by the levels of MHC expression in MSCs. The latter can vary upon MSC inflammatory exposure or differentiation, such as chondrogenic induction, making both priming and differentiation interesting therapeutic strategies. This study investigated the systemic in vivo immune cellular response against allogeneic equine MSCs in these situations. Either MSCs in basal conditions (MSC-naïve), pro-inflammatory primed (MSC-primed) or chondrogenically differentiated (MSC-chondro) were repeatedly administered subcutaneously into autologous, MHC-matched or MHC-mismatched allogeneic equine recipients. At different time-points after each administration, lymphocytes were obtained from recipient horses and exposed in vitro to the same type of MSCs to assess the proliferative response of different T cell subsets (cytotoxic, helper, regulatory), B cells, and interferon gamma (IFNγ) secretion. Higher proliferative response of helper and cytotoxic T lymphocytes and IFNγ secretion was observed in response to all types of MHC-mismatched MSCs over MHC-matched ones. MSC-primed produced the highest immune response, followed by MSC-naïve, and MSC-chondro. However, MSC-primed activated Treg and had a mild effect on B cells, and the response after their second administration was similar to the first one. On the other hand, both MSC-chondro and MSC-naïve barely induced Treg response but promoted B lymphocyte activation, and proportionally induced a higher cell response after the second administration. In conclusion, both the type of MSC conditioning and the MHC compatibility influenced systemic immune recognition of equine MSCs after single and repeated administrations, but the response was different. Selecting MHC-matched donors would be particularly recommended for MSC-primed and repeated MSC-naïve administrations. While MHC-mismatching in MSC-chondro would be less critical, B cell response should not be ignored. Comprehensively investigating the in vivo immune response against equine allogeneic MSCs is crucial for advancing veterinary cell therapies.