Development of an ex vivo organ culture model using human gastro-intestinal tissue and Campylobacter jejuni

Campylobacter jejuni is an important food-borne pathogen. However, relatively little is understood regarding its pathogenesis, and research is hampered by the lack of a suitable model. Recently, a number of groups have developed assays to study the pathogenic mechanisms of C. jejuni using cell culture models. Here, we report the development of an ex vivo organ culture model, allowing for the maintenance of intestinal mucosal tissue, to permit more complex host-bacterium interactions to be studied. Ex vivo organ culture highlights the propensity for C. jejuni to adhere to mucosal tissue via the flagellum, either as discrete colonies or as multicellular units.     

Isolation and ex vivo expansion of synovial mesenchymal stromal cells for cartilage repair

Background aimsHyaline articular cartilage is a highly specialized tissue that offers a low-friction and wear-resistant interface for weight-bearing surface articulation in diarthrodial joints, but it lacks vascularity. It displays an inherent inability to heal when injured in a skeletally mature individual. Joint-preserving treatment procedures such as mosaicplasty, débridement, perichondrium transplantation and autologous chondrocyte implantation have shown variable results, and the average long-term result is sub-standard. Because of these limitations of the treatment methods and lack of intrinsic repair capacity of mature cartilage tissue, an alternative treatment approach is needed, and synovial mesenchymal stromal cells (SMSCs) represent an attractive therapeutic alternative because of their ex vivo proliferation capacity, multipotency and ability to undergo chondrogenesis.MethodsSMSCs were isolated from tissues obtained by arthroscopy using two types of biopsies. Ex vivo cell expansion was accomplished under static and dynamic culture followed by characterization of cells according to the International Society for Cellular Therapy guidelines. Kinetic growth models and metabolite analysis were used for understanding the growth profile of these cells.ResultsFor the first time, SMSCs were expanded in stirred bioreactors and achieved higher cell density in a shorter period of time compared with static culture or with other mesenchymal stromal cell sources.ConclusionsIn this study we were able to achieve (8.8 ± 0.2) × 10⁵ cells within <2 weeks in dynamic culture under complete xeno-free conditions. Our results also provided evidence that after dynamic culture these cells had an up-regulation of chondrogenic genes, which can be a potential factor for articular cartilage regeneration in clinical settings.     

Strategies to enhance immunomodulatory properties and reduce heterogeneity in mesenchymal stromal cells during ex vivo expansion

Therapies using mesenchymal stromal cells (MSCs) to treat immune and inflammatory conditions are now at an exciting stage of development, with many MSC-based products progressing to phase II and III clinical trials. However, a major bottleneck in the clinical translation of allogeneic MSC therapies is the variable immunomodulatory properties of MSC products due to differences in their tissue source, donor heterogeneity and processes involved in manufacturing and banking. This variable functionality of MSC products likely contributes to the substantial inconsistency observed in the clinical outcomes of phase III trials of MSC therapies; several trials have failed to reach the primary efficacy endpoint. In this review, we discuss various strategies to consistently maintain or enhance the immunomodulatory potency of MSCs during ex vivo expansion, which will enable the manufacture of allogeneic MSC banks that have high potency and low variability. Biophysical and biochemical priming strategies, the use of culture additives such as heparan sulfates, and genetic modification can substantially enhance the immunomodulatory properties of MSCs during in vitro expansion. Furthermore, robust donor screening, the use of biomarkers to select for potent MSC subpopulations, and rigorous quality testing to improve the release criteria for MSC banks have the potential to reduce batch-to-batch heterogeneity and enhance the clinical efficacy of the final MSC product. Machine learning approaches to develop predictive models of individual patient response can enable personalized therapies and potentially establish correlations between in vitro potency measurements and clinical outcomes in human trials.     

Invasion of Wolbachia into Anopheles and other insect germlines in an ex vivo organ culture system

The common bacterial endosymbiont Wolbachia manipulates its host's reproduction to promote its own maternal transmission, and can interfere with pathogen development in many insects making it an attractive agent for the control of arthropod-borne disease. However, many important species, including Anopheles mosquitoes, are uninfected. Wolbachia can be artificially transferred between insects in the laboratory but this can be a laborious and sometimes fruitless process. We used a simple ex vivo culturing technique to assess the suitability of Wolbachia-host germline associations. Wolbachia infects the dissected germline tissue of multiple insect species when the host tissue and bacteria are cultured together. Ovary and testis infection occurs in a density-dependent manner. Wolbachia strains are more capable of invading the germline of their native or closely related rather than divergent hosts. The ability of Wolbachia to associate with the germline of novel hosts is crucial for the development of stably-transinfected insect lines. Rapid assessment of the suitability of a strain-host combination prior to transinfection may dictate use of a particular Wolbachia strain. Furthermore, the cultured germline tissues of two major Anopheline vectors of Plasmodium parasites are susceptible to Wolbachia infection. This finding further enhances the prospect of using Wolbachia for the biological control of malaria.     

Effect of cell-seeding density on the proliferation and gene expression profile of human umbilical vein endothelial cells within ex vivo culture

Background aimsCharacterization of endothelial cell–biomaterial interaction is crucial for the development of blood-contacting biomedical devices and implants. However, a crucial parameter that has largely been overlooked is the cell-seeding density.MethodsThis study investigated how varying cell-seeding density influences human umbilical vein endothelial cell (HUVEC) proliferation on three different substrata: gelatin, tissue culture polystyrene (TCPS) and poly-l-lactic acid (PLLA).ResultsThe fastest proliferation was seen on gelatin, followed by TCPS and PLLA, regardless of seeding density. On both TCPS and gelatin, maximal proliferation was attained at an initial seeding density of 1000 cells/cm². At seeding densities above and below 1000 cells/cm², the proliferation rate decreased sharply. On PLLA, there was a decrease in cell numbers over 7 days of culture, below a certain threshold seeding density (c. 2500–3000 cells/cm²), which meant that some of the cells were dying off rather than proliferating. Above this threshold seeding density, HUVEC displayed slow proliferation. Subsequently, quantitative real-time polymerase chain reaction (RT-qPCR) analysis of eight gene markers associated with adhesion and endothelial functionality (VEGF-A, integrin-α5, VWF, ICAM1, ICAM2, VE-cadherin, endoglin and PECAM1) was carried out on HUVEC seeded at varying densities on the three substrata. A significant downregulation of gene expression was observed at an ultralow cell-seeding density of 100 cells/cm². This was accompanied by an extremely slow proliferation rate, probably because of an acute lack of intercellular contacts and paracrine signaling.ConclusionHence, this study demonstrates that seeding density has a profound effect on the proliferation and gene expression profile of endothelial cells seeded on different biomaterial surfaces.     

Effect of cell culture media on extracellular vesicle secretion from mesenchymal stromal cells and neurons

BackgroundExtracellular vesicles (EVs) secreted by neuronal cells in vitro have promising therapeutic potential for brain diseases. Optimization of cell culture conditions and methodologies for high-yield isolation of EVs for preclinical and clinical applications, however, remains a challenge.ObjectiveTo probe the cell culture conditions required for optimal EV secretion by human-derived neuronal cells.MethodologyFirst, we optimized the EV purification protocol using human mesenchymal stromal cell (MSC) cultures. Next, we compared the effects of different variables in human pluripotent stem cell (hPSC)-derived neuronal cultures on EV secretion. EVs were isolated from cell conditioned media (CCM) and control media with no cells (NCC) using ultrafiltration combined with size-exclusion chromatography (SEC). The hPSC neurons were cultured in 2 different media from which EVs were collected at 2 maturation time-points (days 46 and 60). Stimulation with 25 mM KCl was also evaluated as an activator of EV secretion by neurons. The collected SEC fractions were analyzed by nanoparticle tracking analysis (NTA), protein concentration assay, and blinded transmission electron microscopy (TEM).ResultsA peak in cup-shaped particles was observed in SEC fractions 7–10 of MSC samples, but not corresponding media controls, indicating successful isolation of EVs. Culture medium had no significant effect on EV yield. The EV yield of the samples did not differ significantly according to the culture media used or the cell maturation time-points. Stimulation of neurons with KCl for 3 h reduced rather than increased the EV yield.ConclusionsWe demonstrated successful EV isolation from MSC and neuronal cells using an ultrafiltration-SEC method. The EV yield from MSC and neuronal cultures exhibited a large batch effect, apparently related to the culture media used, highlighting the importance of including NCC as a negative control in all cell culture experiments.     

Chondrogenic differentiation of human mesenchymal stem cells within an alginate layer culture system

Human mesenchymal stem cells (hMSCs) derived from bone marrow have the capacity to differentiate along a number of connective tissue pathways and are an attractive source of chondrocyte precursor cells. When these cells are cultured in a three-dimensional format in the presence of transforming growth factor-beta, they undergo characteristic morphological changes concurrent with deposition of cartilaginous extracellular matrix (ECM). In this study, factors influencing hMSC chondrogenesis were investigated using an alginate layer culture system. Application of this system resulted in a more homogeneous and rapid synthesis of cartilaginous ECM than did micromass cultures and presented a more functional format than did alginate bead cultures. Differentiation was found to be dependent on initial cell seeding density and was interrelated to cellular proliferation. Maximal glycosaminoglycan (GAG) synthesis defined an optimal hMSC seeding density for chondrogenesis at 25 x 10(6) cells/ml. Inclusion of hyaluronan in the alginate layer at the initiation of cultures enhanced chondrogenic differentiation in a dose-dependent manner, with maximal effect seen at 100 microg/ml. Hyaluronan increased GAG synthesis at early time points, with greater effect seen at lower cell densities, signifying cell-cell contact involvement. This culture system offers additional opportunities for elucidating conditions influencing chondrogenesis and for modeling cartilage homeostasis or osteoarthritic changes.     

Clonal nature of fibroblast colonies formed by stromal bone marrow cells in culture

The clonal nature of CFUf-derived fibroblast colonies was tested in mixed cultures of CBA and CBAT6T6 bone marrow cells. Inoculation of marrow cell suspensions into flasks coated with poly-I-lysin has proved that no stromal aggregates were present among cells subjected to explantation. Marrow cell cultures depleted of macrophages and myeloid cells were used for chromosome analysis. The coincidence of karyotypes within a stromal colony was found in mixed cultures, which proves that CFUf-derived fibroblast colonies are cell clones.     

Gelation characteristics and osteogenic differentiation of stromal cells in inert hydrolytically degradable micellar polyethylene glycol hydrogels

The use of poly(ethylene glycol) (PEG) hydrogels in tissue engineering is limited by their persistence in the site of regeneration. In an attempt to produce inert hydrolytically degradable PEG-based hydrogels, star (SPELA) poly(ethylene glycol-co-lactide) acrylate macromonomers with short lactide segments (<15 lactides per macromonomer) were synthesized. The SPELA hydrogel was characterized with respect to gelation time, modulus, water content, sol fraction, degradation, and osteogenic differentiation of encapsulated marrow stromal cells (MSCs). The properties of SPELA hydrogel were compared with those of the linear poly(ethylene glycol-co-lactide) acrylate (LPELA). The SPELA hydrogel had higher modulus, lower water content, and lower sol fraction than the LPELA. The shear modulus of SPELA hydrogel was 2.2 times higher than LPELA, whereas the sol fraction of SPELA hydrogel was 5 times lower than LPELA. The degradation of SPELA hydrogel depended strongly on the number of lactide monomers per macromonomer (nL) and showed a biphasic behavior. For example, as nL increased from 0 to 3.4, 6.4, 11.6, and 14.8, mass loss increased from 7 to 37, 80, 100% and then deceased to 87%, respectively, after 6 weeks of incubation. The addition of 3.4 lactides per macromonomer (<10 wt % dry macromonomer or <2 wt % swollen hydrogel) increased mass loss to 50% after 6 weeks. Molecular dynamic simulations demonstrated that the biphasic degradation behavior was related to aggregation and micelle formation of lactide monomers in the macromonomer in aqueous solution. MSCs encapsulated in SPELA hydrogel expressed osteogenic markers Dlx5, Runx2, osteopontin, and osteocalcin and formed a mineralized matrix. The expression of osteogenic markers and extent of mineralization was significantly higher when MSCs were encapsulated in SPELA hydrogel with the addition of bone morphogenetic protein-2 (BMP2). Results demonstrate that hydrolytically degradable PEG-based hydrogels are potentially useful as a delivery matrix for stem cells in regenerative medicine.     

Efficient expansion of mesenchymal stromal cells in a disposable fixed bed culture system

The need for efficient and reliable technologies for clinical‐scale expansion of mesenchymal stromal cells (MSC) has led to the use of disposable bioreactors and culture systems. Here, we evaluate the expansion of cord blood‐derived MSC in a disposable fixed bed culture system. Starting from an initial cell density of 6.0 × 10⁷ cells, after 7 days of culture, it was possible to produce of 4.2(±0.8) × 10⁸ cells, which represents a fold increase of 7.0 (±1.4). After enzymatic retrieval from Fibra‐Cell disks, the cells were able to maintain their potential for differentiation into adipocytes and osteocytes and were positive for many markers common to MSC (CD73, CD90, and CD105). The results obtained in this study demonstrate that MSC can be efficiently expanded in the culture system. This novel approach presents several advantages over the current expansion systems, based on culture flasks or microcarrier‐based spinner flasks and represents a key element for MSC cellular therapy according to GMP compliant clinical‐scale production system. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 29: 568–572, 2013     

Chondroid bone arises from mesenchymal stem cells in organ culture of mandibular condyles

Mandibular condyles of fetal mice 19 to 20 days in utero comprising clean cartilage and its perichondrium were cultured for up to 14 days, and their capacity to develop osteoid and to mineralize in vitro was examined. After 3 days in culture the cartilage of the mandibular condyle appeared to have lost its inherent structural characteristics, including its various cell layers: chondroprogenitor, chondroblastic, and hypertrophic cells. At that time interval no chondroblasts could be seen; instead, most of the cartilage consisted of hypertrophic chondrocytes. By that time, the surrounding perichondrium, which contains pluripotential mesenchymal stem cells, revealed the first signs of extracellular matrix enclosing type I collagen, bone alkaline phosphatase, osteonection, fibronectin, and bone sialoprotein as demonstrated by immunofluorescent techniques. Electron microscopic examinations of the newly formed matrix revealed foci of mineralization within and along collagen fibers as is normally observed during bone development. The composition of the latter mineral deposits resembled calcium pyrophosphate crystals. Following 14 days in culture larger portions of the condyle revealed signs of osseous matrix, yet the tissue reacted positively for type II collagen. Hence, the condylar cartilage, a genuine representative of secondary-type cartilage, elaborated in vitro a unique type of bone that would be most appropriately defined as chondroid bone. Biochemical assays indicated that the de novo formation of chondroid bone was correlated with changes in alkaline phosphatase activity and 45Ca incorporation. The findings of the present study imply that mesenchymal stem cells that ordinarily differentiate into cartilage possess the capacity to differentiate into osteogenic cells and form chondroid bone.     

Successful isolation and ex vivo expansion of human mesenchymal stem/stromal cells obtained from different synovial tissue-derived (biopsy) samples

Mesenchymal stromal cells (MSC) isolated from synovial tissues constitute a novel source of stem-like cells with promising applications in cartilage regeneration and potentially in other regenerative medicine and tissue-engineering settings. Detailed characterization of these cells is lacking, thus compromising their full potential. Here we present the detailed characterization of the ex vivo expansion of synovium-derived stromal cells collected by three different biopsy methods: synovium-direct biopsy, arthroscopic trocar shaver blade filtrate, and cells isolated from synovial fluid (SF) samples. Isolation success rates were >75% for all sources. MSC obtained from the different samples displayed the characteristic immunophenotype of adult MSC, expressing CD73, CD90, and CD105. Arthroscopic shaver blade-derived cells showed the higher proliferation capacity measured by the fold increase (FI) in total cell number over several passages and considering their cumulative population doublings (CPD; 15 ± 0.85 vs. 13 ± 0.73 for synovium vs. 11 ± 0.97 for SF). Also, these cells were able to sustain an increased proliferation under hypoxic (2% O₂) conditions (FI 55 ± 4 vs. 37 ± 7) after 17 days in culture. Expanded cells were able to differentiate successfully along the osteogenic, adipogenic, and chondrogenic lineages in vitro. Overall, these results demonstrate that synovial tissues represent a promising source for the isolation of human MSC, while depicting the variability associated to the biopsy method used, which impact cell behavior in vitro.     

Effects of microgravity simulation on the production of interleukins in culture of human mesenchymal stromal cells

The effects of microgravity that were simulated using clinoritation and cell exposure in a Random Positioning Machine (RPM; Dutch Space, Netherlands) on the production of interleukins by mesenchymal stromal cells (MSCs) of the human bone marrow were studied. Additionally, interleukins production was studied in osteogenic derivatives of MSCs induced by growth factors, such as 10⁻⁸ M dexamethasone, 0.2 mM ascorbic acid, and 10 mM β-glycerolphosphate. Twenty-day clinoritation resulted in an increase in the interleukin-8 (IL-8) level by a factor of 1.4–3.2 in the culture medium of MSCs and their osteogenic derivatives. Exposure of cells to RPM was followed by an increase in IL-8 production by factors of 1.5–6 and 1.6–2.1 after 10 and 20 days of the treatment, respectively. MSCs and their osteogenic derivatives exhibited a tendency towards a decrease in the release of IL-6 into the culture medium after simulation of microgravity with RPM. Thus, in different models, microgravity modified production of interleukins in MSCs and mature cells with the osteogenic phenotype.     

Adenoviral gene transfer of eNOS: high-level expression in ex vivo expanded marrow stromal cells

Endothelial nitric oxide synthase (eNOS) is an attractive target for cardiovascular gene therapy. Marrow stromal cells (MSCs), also known as mesenchymal stem cells, hold great promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated eNOS gene transfer into ex vivo expanded MSCs, rat MSCs (rMSCs) were isolated, expanded ex vivo, and transduced with Ad5RSVeNOS, an adenoviral vector containing the eNOS gene under the control of the Rous sarcoma virus promoter. The presence of eNOS protein in Ad5RSVeNOS-transduced rMSCs was confirmed by immunohistochemical and Western blot analysis. Transduction efficiency was dose dependent, and eNOS transgene expression in rMSCs persisted for =" BORDER="0">21 days in culture. The rMSCs retained multipotential differentiation capability after adenoviral-mediated eNOS gene transfer. Furthermore, intracavernosal injection of Ad5RSVeNOS-transduced rMSCs increased the expression of eNOS in the corpus cavernosum, and stem cells were identified within corporal sinusoids. These findings demonstrate that replication-deficient recombinant adenovirus can be used to engineer ex vivo expanded rMSCs and that high-level eNOS transgene expression can be achieved, pointing out the clinical potential of using this novel adult stem cell-based gene therapy method for the treatment of cardiovascular diseases. adenoviral vector; nitric oxide; gene expression; differentiation; gene therapy     

Efficient ex vivo induction of T cells with potent anti-tumor activity by protein antigen encapsulated in nanoparticles

Protein antigen (Ag)-based immunotherapies have the advantage to induce T cells with a potentially broad repertoire of specificities. However, soluble protein Ag is generally poorly cross-presented in MHC class I molecules and not efficient in inducing robust cytotoxic CD8⁺ T cell responses. In the present study, we have applied poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NP) which strongly improve protein Ag presentation by dendritic cells (DC) in the absence of additional Toll-like receptor ligands or targeting devices. Protein Ag-loaded DC were used as antigen presenting cells to stimulate T cells in vitro and subsequently analyzed in vivo for their anti-tumor effect via adoptive transfer, a treatment strategy widely studied in clinical trials as a therapy against various malignancies. In a direct comparison with soluble protein Ag, we show that DC presentation of protein encapsulated in plain PLGA-NP results in efficient activation of CD4⁺ and CD8⁺ T cells as reflected by high numbers of activated CD69⁺ and CD25⁺, interferon (IFN)-γ and interleukin (IL)-2-producing T cells. Adoptive transfer of PLGA-NP-activated CD8⁺ T cells in tumor-bearing mice displayed good in vivo expansion capacity, potent Ag-specific cytotoxicity and IFN-γ cytokine production, resulting in curing mice with established tumors. We conclude that delivery of protein Ag through encapsulation in plain PLGA-NP is a very efficient and simple procedure to stimulate potent anti-tumor T cells.     

Transplantation of human neonatal foreskin stromal cells in ex vivo organotypic cultures of embryonic chick femurs

We have previously reported that human neonatal foreskin stromal cells (hNSSCs) promote angiogenesis in vitro and in chick embryo chorioallantoic membrane (CAM) assay in vivo. To examine the in vivo relevance of this observation, we examined in the present study the differentiation potential of hNSSCs in ex vivo organotypic cultures of embryonic chick femurs. Isolated embryonic chick femurs (E10 and E11) were cultured for 10 days together with micro-mass cell pellets of hNSSCs, human umbilical vein endothelial cells (HUVEC) or a combination of the two cell types. Changes in femurs gross morphology and integration of the cells within the femurs were investigated using standard histology and immunohistochemistry. After 10 days, the femurs that were cultured in the presence of hNSSCs alone or hNSSC + HUVEC cells grew longer, exhibited thicker diaphysis and an enlarged epiphyseal region compared to control femurs cultured in the absence of cells. Analysis of cell–femur interactions, revealed intense staining for CD31 and enhanced deposition of collagen rich matrix along the periosteum in femurs cultured with hNSSCs alone or hNSSCs + HUVEC and the most pronounced effects were observed in hNSSC + HUVEC cultures. Our data suggest that organotypic cultures can be employed to test the differentiation potential of stem cells and demonstrate the importance of stem cell interaction with 3D-intact tissue microenvironment for their differentiation.