Comparative Evaluation of the In Vitro Cytotoxicity of a Series of Chitosans and Chitooligosaccharides Water-Soluble at Physiological pH.

Chitosans (CS) have been of great interest due to their properties and numerous applications. However, CS have poor solubility in neutral and basic media, which limits their use in these conditions. In contrast, chitooligosaccharides (COS) have better solubility in water and lower viscosity in aqueous solutions whilst maintaining interesting biological properties. CS and COS, unlike other sugars, are not single polymers with a defined structure but are groups of molecules with modifiable structural parameters, allowing the adaptation and optimization of their properties. The great versatility of CS and COS makes these molecules very attractive for different applications, such as cryopreservation. Here, we investigated the effect of the degree of polymerization (DP), degree of N-acetylation (DA) and concentration of a series of synthesized CS and COS, water-soluble at physiological pH, on their cytotoxicity in an L929 fibroblast cell culture. Our results demonstrated that CS and COS showed no sign of toxicity regarding cell viability at low concentrations (≤10 mg/mL), independently of their DP and DA, whereas a compromising effect on cell viability was observed at a high concentration (100 mg/mL).

Contactless mechanical stimulation of tissue engineered constructs: Development and validation of an air-pulse device.

OBJECTIVE: Tissue engineering (TE) is the study and development of biological substitutes to restore, maintain or improve tissue function. Tissue engineered constructs (TECs) still present differences in mechanical and biological properties compared to native tissue. Mechanotransduction is the process through which mechanical stimulation triggers proliferation, apoptosis, and extracellular matrix synthesis, among other cell activities. Regarding that aspect, the effect of in vitro stimulations such as compression, stretching, bending or fluid shear stress loading modalities have been extensively studied. A fluid flow used to produce contactless mechanical stimulation induced by an air pulse could be easily achieved in vivo without altering the tissue integrity. METHODS: A new air-pulse device for contactless and controlled mechanical simulation of a TECs was developed and validated in this study conducted in the following three phases: 1) conception of the controlled air-pulse device combined with a 3D printed bioreactor; 2) experimental and numerical mechanical characterization of the air-pulse impact by digital image correlation; and 3) achieving sterility and noncytotoxicity of the air-pulse and of the 3D printed bioreactor using a novel dedicated sterilization process. RESULTS: We demonstrated that the treated PLA (polylactic acid) was noncytotoxic and did not influence cell proliferation. An ethanol/autoclaved sterilization protocol for 3D printed objects in PLA has been developed in this study, enabling the use of 3D printing in cell culture. A numerical twin of the device was developed and experimentally characterized by digital image correlation. It showed a coefficient of determination R2 = 0.98 between the numerical and averaged experimental surface displacement profiles of the TEC substitute. CONCLUSION: The results of the study assessed the noncytotoxicity of PLA for prototyping by 3D printing the homemade bioreactor. A novel sterilization process for PLA was developed in this study based on a thermochemical process. A numerical twin using fluid-structure interaction method has been developed to investigate the micromechanical effects of air pulses inside the TEC, which cannot all be measured experimentally, for instance, wave propagation generated during the air-pulse impact. The device could be used to study the cell response to contactless cyclic mechanical stimulation, particularly in TEC with fibroblasts, stromal cells and mesenchymal stem cells, which have been shown to be sensitive to the frequency and strain level at the air-liquid interface.

Therapeutic efficacy of 166Holmium siloxane in microbrachytherapy of induced glioblastoma in minipig tumor model.

Glioblastoma is considered the most common malignant primary tumor of central nervous system. In spite of the current standard and multimodal treatment, the prognosis of glioblastoma is poor. For this reason, new therapeutic approaches need to be developed to improve the survival time of the glioblastoma patient. In this study, we performed a preclinical experiment to evaluate therapeutic efficacy of 166Ho microparticle suspension administered by microbrachytherapy on a minipig glioblastoma model. Twelve minipigs were divided in 3 groups. Minipigs had injections into the tumor, containing microparticle suspensions of either 166Ho (group 1; n = 6) or 165Ho (group 2; n = 3) and control group (group 3; n = 3). The survival time from treatment to euthanasia was 66 days with a good state of health of all minipigs in group 1. The median survival time from treatment to tumor related death were 8.6 and 7.3 days in groups 2 and control, respectively. Statistically, the prolonged life of group 1 was significantly different from the two other groups (p < 0.01), and no significant difference was observed between group 2 and control (p=0.09). Our trial on the therapeutic effect of the 166Ho microparticle demonstrated an excellent efficacy in tumor control. The histological and immunohistochemical analysis showed that the efficacy was related to a severe 166Ho induced necrosis combined with an immune response due to the presence of the radioactive microparticles inside the tumors. The absence of reflux following the injections confirms the safety of the injection device.

Feasibility of intratumoral 165Holmium siloxane delivery to induced U87 glioblastoma in a large animal model, the Yucatan minipig.

Glioblastoma is the most aggressive primary brain tumor leading to death in most of patients. It comprises almost 50-55% of all gliomas with an incidence rate of 2-3 per 100,000. Despite its rarity, overall mortality of glioblastoma is comparable to the most frequent tumors. The current standard treatment combines surgical resection, radiotherapy and chemotherapy with temozolomide. In spite of this aggressive multimodality protocol, prognosis of glioblastoma is poor and the median survival remains about 12-14.5 months. In this regard, new therapeutic approaches should be developed to improve the life quality and survival time of the patient after the initial diagnosis. Before switching to clinical trials in humans, all innovative therapeutic methods must be studied first on a relevant animal model in preclinical settings. In this regard, we validated the feasibility of intratumoral delivery of a holmium (Ho) microparticle suspension to an induced U87 glioblastoma model. Among the different radioactive beta emitters, 166Ho emits high-energy ß(-) radiation and low-energy γ radiation. ß(-) radiation is an effective means for tumor destruction and γ rays are well suited for imaging (SPECT) and consequent dosimetry. In addition, the paramagnetic Ho nucleus is a good asset to perform MRI imaging. In this study, five minipigs, implanted with our glioblastoma model were used to test the injectability of 165Ho (stable) using a bespoke injector and needle. The suspension was produced in the form of Ho microparticles and injected inside the tumor by a technique known as microbrachytherapy using a stereotactic system. At the end of this trial, it was found that the 165Ho suspension can be injected successfully inside the tumor with absence or minimal traces of Ho reflux after the injections. This injection technique and the use of the 165Ho suspension needs to be further assessed with radioactive 166Ho in future studies.

Development of induced glioblastoma by implantation of a human xenograft in Yucatan minipig as a large animal model.

BACKGROUND: Glioblastoma is the most common and deadliest primary brain tumor for humans. Despite many efforts toward the improvement of therapeutic methods, prognosis is poor and the disease remains incurable with a median survival of 12-14.5 months after an optimal treatment. To develop novel treatment modalities for this fatal disease, new devices must be tested on an ideal animal model before performing clinical trials in humans. NEW METHOD: A new model of induced glioblastoma in Yucatan minipigs was developed. Nine immunosuppressed minipigs were implanted with the U87 human glioblastoma cell line in both the left and right hemispheres. Computed tomography (CT) acquisitions were performed once a week to monitor tumor growth. RESULTS: Among the 9 implanted animals, 8 minipigs showed significant macroscopic tumors on CT acquisitions. Histological examination of the brain after euthanasia confirmed the CT imaging findings with the presence of an undifferentiated glioma. COMPARISON WITH EXISTING METHOD: Yucatan minipig, given its brain size and anatomy (gyrencephalic structure) which are comparable to humans, provides a reliable brain tumor model for preclinical studies of different therapeutic METHODS: in realistic conditions. Moreover, the short development time, the lower cyclosporine and caring cost and the compatibility with the size of commercialized stereotactic frames make it an affordable and practical animal model, especially in comparison with large breed pigs. CONCLUSION: This reproducible glioma model could simulate human anatomical conditions in preclinical studies and facilitate the improvement of novel therapeutic devices, designed at the human scale from the outset.

Characterization of a novel canine T-cell line established from a spontaneously occurring aggressive T-cell lymphoma with large granular cell morphology.

Dogs with lymphoma are established as good model for human non-Hodgkin lymphoma studies. Canine cell lines derived from lymphomas may be valuable tools for testing new therapeutic drugs. In this context, we established a canine T-cell line, PER-VAS, from a primary aggressive T-cell lymphoma with large granular morphology. Flow cytometric analysis revealed a stable immunophenotype: PER-VAS cells were positively labelled for CD5, CD45, MHC II and TLR3, and were negative for CD3, CD4 and CD8 expression. Although unstable along the culture process, IL-17 and MMP12 proteins were detectable as late as at passages 280 and 325i.e. respectively 24 and 29 months post isolation. At passage 325, PER-VAS cells maintained the expression of IL-17, CD3, CD56, IFNγ and TNFα mRNAs as shown by RT-PCR analysis. Stable rearrangement of the TCRγ gene has been evidenced by PCR. PER-VAS cells have a high proliferation index with a doubling time of 16.5h and were tumorigenic in Nude mice. Compared to the canine cell lines already reported, PER-VAS cells display an original expression pattern, close to NKT cells, which makes them valuable tools for in vitro comparative research on lymphomas.

Toll-like receptor 3 (TLR3): a new marker of canine monocytes-derived dendritic cells (cMo-DC).

Toll-like receptors (TLRs) are a family of functionally important receptors for recognition of pathogen-associated molecular pattern (PAMP) since they trigger the pro-inflammatory response and upregulation of costimulatory molecules, linking the rapid innate response to adaptative immunity. In human leukocytes, TLR3 has been found to be specifically expressed in dendritic cells (DC). This study examined the expression of TLR3 in canine monocytes-derived DC (cMo-DC) and PBMC using three new anti-TLR3 mAbs (619F7, 722E2 and 713E4 clones). The non-adherent cMo-DC generated after culture in canine IL-4 plus canine GM-CSF were labelled with the three anti-TLR3 clones by flow cytometry, with a strong expression shown for 619F7 and 722E2 clones. By contrast, TLR3 expression was low to moderate in canine monocytes and lymphocytes. These results were confirmed by Western blot using 619F7 and 722E2 clones and several polypeptide bands were observed, suggesting a possible cleavage of TLR3 molecule or different glycosylation states. In addition, TLR3 was detectable in immunocytochemistry by using 722E2 clone. In conclusion, this first approach to study canine TLR3 protein expression shows that three anti-TLR3 clones detect canine TLR3 and can be used to better characterize canine DC and the immune system of dogs.

CD86 molecule is a specific marker for canine monocyte-derived dendritic cells.

In this study, canine monocyte-derived dendritic cells (cMo-DC) were produced in presence of canine GM-CSF (cGM-CSF) and canine IL-4 (cIL-4), and they were characterized by their dendritic morphology, MLR functionality and phenotype. We noticed that cMo-DC were labelled with three anti-human CD86 (FUN-1, BU63 and IT2.2 clones), whereas resting and activated lymphocytes or monocytes were not stained. CD86 expression was induced by cIL-4 and was up-regulated during the differentiation of the cMo-DC, with a maximum at day 7. Furthermore, cMo-DC were very potent even in low numbers as stimulator cells in allogeneic MLR, and BU63 mAb was able to completely block the cMo-DC-induced proliferation in MLR. We also observed that cMo-DC highly expressed MHC Class II and CD32, but we failed to determine their maturation state since the lack of commercially available canine markers. Moreover, cMo-DC contained cytoplasmic periodic microstructures, potentially new ultrastructural markers of canine DC recently described. In conclusion, this work demonstrates that the CD86 costimulatory marker is now usable for a better characterization of in vitro canine DC.

Evaluation of elutriation and magnetic microbead purification of canine monocytes.

An elutriation technique was developed to obtain large quantities of pure canine monocytes. Firstly, peripheral blood mononuclear cells (PBMC) were isolated from whole blood by Ficoll gradient. Then, the PBMC were separated by an elutriation procedure. We demonstrated that these techniques allow the isolation of canine peripheral blood monocytes with a purity of 64% +/- 7.9 when labelled with anti-CD14 antibody. This purity increased to 83% +/- 2.2 after separation by magnetic anti-CD14 microbeads. The cell viability was more than 95% and apoptotic cells were less than 10%. The monocytes purified by these methods were functionally active in a mixed leukocyte reaction (MLR). A lymphocyte fraction was obtained directly only by elutriation with an average of 79.9% +/- 10.7 of CD5+, 7.9% +/- 3.5 of CD21+ and 1.78% +/- 2.53 of CD14+. Our results indicate that this elutriation procedure is a safe method to purify monocytes as well as lymphocytes, useful in MLR.