Lab-on-Chip for Testing Myelotoxic Effect of Drugs and Chemicals

This paper was presented at the 4th Micro and Nano Flows Conference (MNF2014), which was held at University College, London, UK. The conference was organised by Brunel University and supported by the Italian Union of Thermofluiddynamics, IPEM, the Process Intensification Network, the Institution of Mechanical Engineers, the Heat Transfer Society, HEXAG - the Heat Exchange Action Group, and the Energy Institute, ASME Press, LCN London Centre for Nanotechnology, UCL University College London, UCL Engineering, the International NanoScience Community, www.nanopaprika.eu.In the last twenty years, one of the main goals in the drug discovery field has been the development of reliable in vitro models. In particular, in 2006 the European Centre for the Validation of Alternative Methods (ECVAM) has approved the Colony forming Unit-Granulocytes-Macrophages (CFU-GM) test, which is the first and currently unique test applied to evaluate the myelotoxicity of xenobiotics in vitro. The present work aimed at miniaturizing this in vitro assay by developing and validating a Lab-on-Chip (LoC) platform consisting of a high number of bioreactor chambers with screening capabilities in a high-throughput regime

Uptake-release by MSCs of a cationic platinum(II) complex active in vitro on human malignant cancer cell lines

In this study, the in vitro stability of cisplatin (CisPt) and cationic platinum(II)-complex (caPt(II)-complex) and their in vitro activity (antiproliferative and anti-angiogenic properties) were investigated against three aggressive human tumor cell lines. caPt(II)-complex shown a high stability until 9 days of treatment and displayed a significant and higher activity than CisPt against both NCI-H28 mesothelioma (19.37 \ub1 9.57 \u3bcM versus 34.66 \ub1 7.65 \u3bcM for CisPt) and U87 MG glioblastoma (19.85 \ub1 0.97 \u3bcM versus 54.14 \ub1 3.19 for CisPt). Mesenchymal Stromal Cells (AT-MSCs) showed a significant different sensitivity (IC50=71.9 \ub1 15.1 \u3bcM for caPt(II)-complex and 8.7 \ub1 4.5 \u3bcM for CisPt) to the antiproliferative activity of caPt(II)-complex and CisPt. The ability of MSCs to uptake both the drugs in a similar amount of 2.49 pM /cell, suggested a possible development of new therapies based on cell mediated drug delivery

Human Olfactory Bulb Neural Stem Cells (Hu-OBNSCs) Can Be Loaded with Paclitaxel and Used to Inhibit Glioblastoma Cell Growth

Exploitation of the potential ability of human olfactory bulb (hOB) cells to carry, release, and deliver an effective, targeted anticancer therapy within the central nervous system (CNS) milieu remains elusive. Previous studies have demonstrated the marked ability of several types of stem cells (such as mesenchymal stem cells (MSCs) to carry and release different anti-cancer agents such as paclitaxel (PTX). Herein we investigate the ability of human olfactory bulb neural stem cells (Hu-OBNSCs) to carry and release paclitaxel, producing effective cytotoxic effects against cancer cells. We isolated Hu-OBNSCs from the hOB, uploaded them with PTX, and studied their potential cytotoxic effects against cancer cells in vitro. Interestingly, the Hu-OBNSCs displayed a five-fold increase in their resistance to the cytotoxicity of PTX, and the PTX-uploaded Hu-OBNSCs were able to inhibit proliferation and invasion, and to trigger marked cytotoxic effects on glioblastoma multiforme (GBM) cancer cells, and Human Caucasian fetal pancreatic adenocarcinoma 1 (CFPAC-1) in vitro. Despite their ability to resist the cytotoxic activity of PTX, the mechanism by which Hu-OBNSCs acquire resistance to PTX is not yet explained. Collectively our data indicate the ability of the Hu-OBNSCs to resist PTX, and to trigger effective cytotoxic effects against GBM cancer cells and CFPAC-1. This indicates their potential to be used as a carrier/vehicle for targeted anti-cancer therapy within the CNS

Sensitivity of mesenchymal stromal cells to a new imidazole-based cationic Pt(II) complex with high in vitro anticancer activity

OBJECTIVE: Platinum drugs endowed with a novel chemical structure could offer an alternative therapeutic strategy, allowing to enlarge the spectrum of activity and to overcome the many drawbacks of the well-known cisplatin (CisPt) and its derivatives. Our group synthesised a new caPt(II)-complex that showed a very effective cytotoxic effect on triple-negative breast cancer cells and on cell lines partially resistant to cisplatin. In this study, we compared the in vitro stability of CisPt and caPt(II)-complex and their in vitro activity against human tumour cell lines. The drug sensitivity of Mesenchymal Stromal Cells (MSCs) and their ability to uptake and release the drugs was also investigated. MATERIALS AND METHODS: AT-MSCs were isolated, characterized and expanded from human adipose tissue. Drug stability was studied following incubation at 37\ub0C in complete cell culture medium both in the absence and in the presence of a monolayer of MSCs. The effect of CisPt and caPt(II)-complex was tested against mesothelioma (NCI-H28), glioblastoma (U87MG), pancreatic adenocarcinoma (CFPAC-1) and AT-MSCs by using a MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium) anti-proliferative assay in 96 multiwell plates. The amount of drugs incorporated and released by AT-MSCs drugs was evaluated by inductively coupled plasma mass spectrometry (ICP-MS). RESULTS: We found that caPt(II)-complex had a high stability until 9 days of treatment while CisPt lost its anticancer activity after only 24 hours of treatment. CisPt was significantly more active (IC50= 9.64 \ub1 5.10 \ub5M) than caPt(II)-complex (IC50= 21.25 \ub1 6.68 \ub5M) on CFPAC1 proliferation. On the contrary, caPt(II)-complex showed a significant higher activity than CisPt both against NCI-H28 mesothelioma (19.37 \ub1 9.57 \ub5M versus 34.66 \ub1 7.65 \ub5M for CisPt) and U87 MG (19.85 \ub1 0.97 \ub5M versus 54.14 \ub1 3.19 for CisPt). AT-MSCs showed a sensitivity to the cytotoxic effect of caPt(II)-complex (IC50=92.8 \ub1 28.9 \ub5M) and CisPt (IC50= 93.5 \ub1 47.6 \ub5M) that does not differ significantly but with a higher variability of response to CisPt expressed by different donors of AT-MSCs. To the antiproliferative activity of caPt(II)-complex and CisPt, AT-MSCs showed a significant different sensitivity (IC50= 71.9 \ub1 15.1 \ub5M for caPt(II)-complex and 8.7 \ub1 4.5 \ub5M for CisPt). AT-MSCs are able to uptake both the drugs in a similar amount of 2.49 pM /cell. DISCUSSION AND CONCLUSION: The high stability of caPt(II)-complex together with its significant anticancer activity against mesothelioma and glioblastoma makes this new platinum derivative a very interesting molecule able to improve cancer chemotherapy. The low sensitivity of AT- MSCs to the antiproliferative action exerted by caPt(II)-complex together with their ability to uptake and release the drug will be further investigated in order to optimize the drug loading procedure and verify the possibility to set up a system of cell mediated delivery of caPt(II) complex

Non-enzymatic automated closed-cycle process to isolate mesenchymal stromal cells for drug delivery applications

Mesenchymal stromal cells (MSCs) can be easily isolated from several human organs and tissues and, because of their self-renewing capacity and multipotent differentiation properties; they are important tools for treating immune disorder and for tissue repair. To ensure reproducibility, efficacy and safety for clinical uses, these procedures have to be compliant with Good Manufacturing Practices (GMP). Techniques for harvesting and processing human adipose tissue have rapidly evolved in the last years, and, among them, Lipogems represents an innovative approach to obtain micro-fragmented adipose tissue in a short time, without expansion and/or enzymatic treatment. The aim of this study was to assess the presence of Adipose Tissue Mesenchymal Stromal Cells (AT-MSCs) in the drain bag of the device after the standard processing, and the possibility to use these cells as drug delivery agents. We used a Lipogems Processor Prototype (PLG-P) to wash and process the lipoaspirate in a standardized condition. The waste fluid was analyses for the presence of mesenchymal stromal cells that were expanded in flask , characterized by CD analysis and differentiation ability. The ability of these cells to uptake Paclitaxel was evaluated by using a previous standardized procedure. The activity of paclitaxel released by drug loaded AT MSCs were tested in vitro against cancer cells. We found that the drain bag contain red blood cells and oil residues but also significant amount of single isolated cells easy to expand and having the typical characteristics of AT-MSCs. These AT-MSCS were also able to uptake Paclitaxel and then release it in amount active in vitro against the proliferation of a human pancreatic cancer cell line. Our findings suggest the possibility to develop a new integrated in-line device, implying neither enzymatic treatment, nor centrifugation, which can isolate and expand AT-MSCs in an intrinsically closed system, highly simplifying GMP compliance effort. This system could be used to obtain AT-MSCs not only for regenerative purposes, but also for drug loading procedure, as a new and innovative cell mediated drug-delivery system

Mesenchymal Stromal Cells Uptake And Release Paclitaxel Without Reducing Its Anticancer Activity

To improve the drug delivery efficiency on target cells many strategies have been developed including also Mesenchymal Stromal Cells (MSCs) approaches. In a previous study, we found that bone-marrow-derived MSCs (BM-MSCs) were able to incorporate and release the anti-tumor and anti-angiogenic drug Paclitaxel (PTX). In this study, we evaluated the stability of PTX in standard cell culture conditions by analysing the metabolites produced by MSCs after their incorporation of the drug. We are able to show that MSCs do not release neither 3-OH-PTX nor 6-OH-PTX metabolites (having a lower anticancer activity) but the release of an active PTX molecule together with the isomer 7-Epitaxol, known to maintain the whole biological activity. This confirms that the simple procedure of MSCs priming with a drug (without any genetic cell manipulation), in our case PTX, does not modify the activity of the molecule and provide a new biological-device to carry and delivery PTX in tumor sites, by contributing to improve drug efficacy and target selectivity in cancer therapy

Biotechnological applications of Echinoderm mutable collagenous tissues

Echinoderms possess unique connective tissues (Mutable Collagenous Tissues, MCT) which display striking passive mechanical properties. These tissues are widespread in all the five extant echinoderm classes and are fundamental for many aspects of their biology. Besides their evident physiological relevance, MCT can also be a promising source of inspiration and material, namely collagen, for biotechnological applications, as an alternative to the widely used mammalian sources. Collagen-made materials are the most promising in regenerative medicine: in the field of Guided Tissue Regeneration collagen barrier-membranes of mammalian origin (BM) are typically used to facilitate proper tissue regrowth in anatomically separated compartments. Recently, we have proposed the sea urchin peristomial membrane as a target MCT for the extraction of native collagen fibrils and the development of innovative membranes (Di Benedetto et al., 2014). In the present work we used and compared MCT from different echinoderm models, representative of the diverse classes (sea urchin, starfish and sea cucumber) to prepare echinoderm-derived membranes (EDM). The EDM were characterized and compared in terms of structure, biomechanics and human cell behaviour (skin-derived fibroblasts). BM or substrates made of soluble or re-fibrillated bovine collagen were used as further comparison. All the EDM were similar in terms of structure (highly dense fibrillar network) and mechanical performances, but all of them were much thinner (~20-30 folds) and mechanically more resistant (~20 folds) than the commercially used BM. Fibroblasts seeded on the EDM were generally less numerous than those on mammalian collagen substrates (except in the case of the sea urchin-derived collagen membranes, which were similar) and displayed a more elongated shape, less numerous filopodial processes and a different cytoskeletal organization, all these features suggesting a weaker cell attachment. Overall, our data indicate that all the echinoderm models can be considered potential alternative source of collagen to produce membranes suitable for Guided Tissue Regeneration, where high mechanical resistance, low cell attachment and dense fibrillar network for a barrier-effect are required. Nevertheless, sea urchins might display some advantage, also in terms of eco-sustainability (by recycling tissues from food wastes). Further in vivo tests are necessary to confirm the validity of this innovative marine biomaterial. References Di Benedetto C, Barbaglio A, Martinello T, Alongi V, Fassini D, Cullor\ue0 E, Patruno M, Bonasoro F, Barbosa MA, Candia Carnevali MD, Sugni M. 2014. Production, Characterization and Biocompatibility of Marine Collagen Matrices from an Alternative and Sustainable Source: The Sea Urchin Paracentrotus lividus. Marine Drugs. 12: 4912-4933

In vitro inhibition of human mesothelioma cells by paclitaxel-releasing mesenchymal stromal cells

OBJECTIVE: Malignant pleural mesothelioma (MPM) is a rare fatal asbestos-related malignancy originating in the mesothelial cells of the pleura. A platinum-based doublet containing a third-generation antifolate is the front-line standard of care whilst there are no approved second-line treatments for MPM which remains a disease setting to test the efficacy of new therapeutic agents. Recent studies have demonstrated that mesenchymal stromal cells (MSCs) are able to migrate specifically to tumors and their metastatic sites when administered intravenously. Our group previously demonstrated that PTX-primed MSCs provide a new approach for cancer therapy. It also has been reported that a patient with MPM not responsive to standard first-line treatment, had good response after treatment with nanoparticle albumin-bound paclitaxel and carboplatin, suggesting that new delivery strategies might improve the clinical management of this disease. Aim of this study was to evaluate the in vitro antiproliferative effect of PTX-releasing MSCs on human MPM. MATERIALS AND METHODS: Bone marrow mesenchymal stromal cells (BM-MSCs) were loaded with pemetrexed (PMX) and paclitaxel (PTX) according to a standardized procedure. The primed cells (BM-MSCs/PMX and BM-MSCs/PTX) were lysed and tested in vitro by a 7 days antiproliferation MTT assay against NCI-H28 mesothelioma and a panel of tumor cell lines: T98G (glioblastoma multiforme), U87MG (likely glioblastoma), UPCI-SCC-154 (squamous oral carcinoma) and MOLT-4 (acute lymphoblastic leukemia). RESULTS: The in vitro anticancer activity of pure PTX was significantly higher than that of PMX against all the cell lines tested; in particular, on NCI-H28, the activity of PTX was 14.7 times higher than that of PMX. No inhibitory activity was exerted by the lysate of BM-MSCs loaded with PMX (BM-MSCs/PMX), whereas a significant antitumor activity was produced by the lysate from PTX-loaded BM-MSCs (BM-MSCs/ PTX). Based on these data, we calculated that a single drug loaded BM-MSCs cell can delivery about 0.15 pg of PTX. By homing 106 BM-MSC/PTX into 1 cm3 of tumor mass, we can estimate a PTX delivery near to the concentration of 150 ng/ml corresponding to a value 26 times higher than IC50. CONCLUSION: These preliminary results demonstrated the good activity of PTX against a mesothelioma cell line growth in vitro. Furthermore, also PTX-loaded mesenchymal stromal cells can successfully inhibit the in vitro proliferation of human mesothelioma cells. Further studies and in vivo testing are required to confirm these data that could open the way to improve the mesothelioma therapy by apply a cell mediated system for drug delivery

Inhibition of Human Malignant Pleural Mesothelioma Growth by Mesenchymal Stromal Cells

BACKGROUND: Malignant Pleural Mesothelioma (MPM) is an aggressive tumor that has a significant incidence related to asbestos exposure with no effective therapy and poor prognosis. The role of mesenchymal stromal cells (MSCs) in cancer is controversial due to their opposite effects on tumor growth and in particular, only a few data are reported on MSCs and MPM. METHODS: We investigated the in vitro efficacy of adipose tissue-derived MSCs, their lysates and secretome against different MPM cell lines. After large-scale production of MSCs in a bioreactor, their efficacy was also evaluated on a human MPM xenograft in mice. RESULTS: MSCs, their lysate and secretome inhibited MPM cell proliferation in vitro with S or G0/G1 arrest of the cell cycle, respectively. MSC lysate induced cell death by apoptosis. The efficacy of MSC was confirmed in vivo by a significant inhibition of tumor growth, similar to that produced by systemic administration of paclitaxel. Interestingly, no tumor progression was observed after the last MSC treatment, while tumors started to grow again after stopping chemotherapeutic treatment. CONCLUSIONS: These data demonstrated for the first time that MSCs, both through paracrine and cell-to-cell interaction mechanisms, induced a significant inhibition of human mesothelioma growth. Since the prognosis for MPM patients is poor and the options of care are limited to chemotherapy, MSCs could provide a potential new therapeutic approach for this malignancy