Long-term Culture Techniques Research Articles

Small extracellular vesicles derived from four dimensional-culture of mesenchymal stem cells induce alternatively activated macrophages by upregulating IGFBP2/EGFR to attenuate inflammation in the spinal cord injury of rats.

Effectively reducing the inflammatory response after spinal cord injury (SCI) is a challenging clinical problem and the subject of active investigation. This study employed a porous scaffold-based three dimensional long-term culture technique to obtain human umbilical cord mesenchymal stem cell (hUC-MSC)-derived Small Extracellular Vesicles (sEVs) (three dimensional culture over time, the "4D-sEVs"). Moreover, the vesicle size, number, and inner protein concentrations of the MSC 4D-sEVs contained altered protein profiles compared with those derived from 2D culture conditions. A proteomics analysis suggested broad changes, especially significant upregulation of Epidermal Growth Factors Receptor (EGFR) and Insulin-like Growth Factor Binding Protein 2 (IGFBP2) in 4D-sEVs compared with 2D-sEVs. The endocytosis of 4D-sEVs allowed for the binding of EGFR and IGFBP2, leading to downstream STAT3 phosphorylation and IL-10 secretion and effective induction of macrophages/microglia polarization from the pro-inflammatory M1 to anti-inflammatory M2 phenotype, both in vitro and in the injured areas of rats with compressive/contusive SCI. The reduction in neuroinflammation after 4D-sEVs delivery to the injury site epicenter led to significant neuroprotection, as evidenced by the number of surviving spinal neurons. Therefore, applying this novel 4D culture-derived Small Extracellular Vesicles could effectively curb the inflammatory response and increase tissue repair after SCI.

Population genomics in neglected malaria parasites.

Malaria elimination includes neglected human malaria parasites Plasmodium vivax, Plasmodium ovale spp., and Plasmodium malariae. Biological features such as association with low-density infection and the formation of hypnozoites responsible for relapse make their elimination challenging. Studies on these parasites rely primarily on clinical samples due to the lack of long-term culture techniques. With improved methods to enrich parasite DNA from clinical samples, whole-genome sequencing of the neglected malaria parasites has gained increasing popularity. Population genomics of more than 2200 P. vivax global isolates has improved our knowledge of parasite biology and host-parasite interactions, identified vaccine targets and potential drug resistance markers, and provided a new way to track parasite migration and introduction and monitor the evolutionary response of local populations to elimination efforts. Here, we review advances in population genomics for neglected malaria parasites, discuss how the rich genomic information is being used to understand parasite biology and epidemiology, and explore opportunities for the applications of malaria genomic data in malaria elimination practice.

Viral-specific adoptive immunotherapy after allo-SCT: the role of multimer-based selection strategies

Recipients of hematopoietic SCT undergo a period of profound immunosuppression due to the chemotherapy and/or radiotherapy used for the conditioning and to the graft versus host reaction. SCT patients are highly susceptible to the development of viral infections such as CMV or EBV. The achievement of a competent immunological response, such as viral-specific T cells, is associated with a lower incidence of viral infections. Methods for direct identification of antigen-specific T cells have been based on the functional characteristics of these T cells. Techniques such as proliferation and ELISPOT assays, intracellular cytokine staining and IFN-γ capture have been used to quantitate and obtain viral-specific T cells. Multimers are composed of several MHC molecules loaded with immunodominant peptides joined to a fluorescent molecule, which signal can be quantified by a flow cytometer. Multimer technology together with recent advances in flow cytometry, have facilitated the monitoring and selection of antigen-specific T cells without the need for in vitro cultures and manipulation. This has resulted in a better characterization of the function and phenotype of the different subpopulations of T cells involved in the immune recovery post allogeneic SCT. It is becoming a distinct possibility to isolate individual antigen-specific T cells, without long-term culture techniques, and potentially use them as adoptive immunotherapy in the SCT setting.

Improved methods for axenic culture of Labyrinthula terrestris, causal agent of rapid blight of turfgrasses

The genus Labyrinthula is a group of unicellular microorganisms with spindle-shaped cells that move in an ectoplasmic network. Most Labyrinthula species are saprotrophic and found in coastal marine or estuarine habitats; however, exceptions exist, such as Labyrinthula terrestris , a terrestrial plant pathogen that causes rapid blight on cool-season turfgrasses. Labyrinthula spp. can be grown in culture, which facilitates studies on their biology and pathology. However, axenic culture of L.terrestris has always been challenging. We modified the most commonly used Labyrinthula growth medium, serum seawater agar (SSA), and designed 2 media for improved pure culture, modified SSA (MSSA) and grass extract SSA (GESSA). A comparative assessment of these 2 media and basic SSA was made to measure the growth responses of 18 L.terrestris isolates. Results indicate that the average colony area was greatest on GESSA followed by MSSA, while cultures lived longest on MSSA followed by GESSA. We also suggest an improved long-term culture technique to maintain viable L.terrestris isolates for at least 2 years.

Combination of the Hedgehog Pathway Inhibitor LDE225 and Nilotinib Eliminates Chronic Myeloid Leukemia Stem and Progenitor Cells.

Abstract 1428Poster Board I-451Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder arising in a hematopoietic stem cell (HSC). CML is associated with expression of the Philadelphia chromosome (Ph) and its fusion gene product, BCR-ABL, a constitutively active tyrosine kinase. The tyrosine kinase inhibitors (TKIs) imatinib, nilotinib and dasatinib, all show impressive rates of complete cytogenetic response in chronic phase (CP) CML. However, the majority of responding CP CML patients have detectable BCR-ABL transcripts which might arise from a population of quiescent CML stem cells not effectively targeted by TKIs. Recent studies have indicated that the Hedgehog (Hh) pathway, a developmental pathway with roles in primitive and adult hematopoiesis, is activated in CML stem cells via upregulation of Smoothened (SMO), a seven-transmembrane domain receptor protein (Dierks et al, Cancer Cell 2008;14:238). We found that Gli1, a downstream target of Hh signalling, is significantly upregulated (6-fold) at the mRNA level in CD34+ enriched CP CML cells compared to normal CD34+ hematopoietic cells by Taqman quantitative RT-PCR. Thus, inhibition of SMO may be an effective therapeutic strategy to reduce the quiescent CML stem cell pool. LDE225 (Novartis Pharma) is a small molecule SMO antagonist which has recently entered Phase 1 clinical evaluation in patients with solid tumors. We assessed the efficacy of LDE225, alone and in combination with nilotinib, in primary CD34+ CML cells in vitro using short- and long-term culture techniques, including colony forming cell (CFC) assays with replates, long-term culture-initiating cell (LTC-IC) assays, CFSE-based flow cytometry to track cell division, Annexin/Viaprobe to measure apoptosis and Ki-67/7AAD cell cycle analysis. In short-term culture, incremental concentrations of LDE225 (0.5 - 1000nM) had no effect on total viable cell counts up to 12 days and did not inhibit cell proliferation as assessed by BrDU incorporation and CFSE staining. LDE225 did not increase apoptosis or alter the cell cycle profile of CD34+ CML cells compared to untreated controls. The lack of response in short-term culture experiments is explained by the stem cell selective nature of LDE225 which affects self-renewal and not proliferation or apoptosis. For long-term culture experiments, CD34+ CML cells were cultured for either 3 or 7 days in serum-free media supplemented with physiological growth factors (IL-3, IL-6, G-CSF, FLT-3 and SCF) with and without LDE225; cells were then washed and put into CFC assays with no added LDE225. Increasing concentrations of LDE225 did not significantly reduce colony read-out in the CFC assays. However, when the CFCs were serially replated there was a highly significant reduction in secondary colony formation and replating efficiency with increasing concentrations of LDE225 up to a plateau at 50nM during the initial 72 hour culture. In the first re-plate, colony formation was reduced by 38% with 5nM (P=0.07) and 62% with 50nM LDE225 (P=0.011; n=3) compared to a “no drug” control. Further reductions in colony formation were seen in second and subsequent replates. In further CFC replating experiments, after 3 and 7 days initial exposure to the combination of LDE225 10nM + nilotinib 5μM, replating efficiency was reduced by 50% (P<0.03) and 74%,(P<0.005) respectively (n=3). Single agent nilotinib resulted in a non-significant increase in colony formation and replating efficiency. In LTC-IC assays, compared to the “no drug” control, CD34+ CML cells showed increased colony formation in the nilotinib arm, indicating that, by inhibiting proliferation, nilotinib exerts a protective effect on CML stem cells (as shown previously for imatinib and dasatinib; Copland et al, Blood 2008;111:2843). The arm containing LDE225 + nilotinib showed a reduction in colony formation compared to both the nilotinib arm (85% reduction) and the “no drug” control (50% reduction). These results confirm the stem cell selectivity of LDE225. In conclusion, LDE225 targets CML stem cells and, in combination with nilotinib, represents a novel therapeutic strategy for targeting both the primitive quiescent CML stem cell population and the bulk CML progenitor population. In vivo studies in murine models of CML are ongoing to help further evaluate the clinical potential of LDE225 in combination with nilotinib. DisclosuresHolyoake: Bristol-Myers Squibb: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Dorsch: Novartis: Employment. Manley: Novartis: Employment. Bhatia: Novartis: Consultancy. Copland: Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Enamel-free teeth: Tbx1 deletion affects amelogenesis in rodent incisors

TBX1 is a principal candidate gene for DiGeorge syndrome, a developmental anomaly that affects the heart, thymus, parathyroid, face, and teeth. A mouse model carrying a deletion in a functional region of the Tbx1 gene has been extensively used to study anomalies related to this syndrome. We have used the Tbx1 null mouse to understand the tooth phenotype reported in patients afflicted by DiGeorge syndrome. Because of the early lethality of the Tbx1−/− mice, we used long-term culture techniques that allow the unharmed growth of incisors until their full maturity. All cultured incisors of Tbx1−/− mice were hypoplastic and lacked enamel, while thorough histological examinations demonstrated the complete absence of ameloblasts. The absence of enamel is preceded by a decrease in proliferation of the ameloblast precursor cells and a reduction in amelogenin gene expression. The cervical loop area of the incisor, which contains the niche for the epithelial stem cells, was either severely reduced or completely missing in mutant incisors. In contrast, ectopic expression of Tbx1 was observed in incisors from mice with upregulated Fibroblast Growth Factor signalling and was closely linked to ectopic enamel formation and deposition in these incisors. These results demonstrate that Tbx1 is essential for the maintenance of ameloblast progenitor cells in rodent incisors and that its deletion results in the absence of enamel formation.

Long-term culture of adult murine epidermal keratinocytes

Long-term cultures of epidermal cells from mouse skin have been notoriously difficult to establish. To develop a modified serum-free medium and technique for long-term culture of adult mouse epidermal keratinocytes. Epidermal cells from trypsin-treated adult mouse dorsal and ventral skin were grown on type I collagen-coated dishes without feeder layers in a serum-free medium supplemented with only 10 ng mL(-1) epidermal growth factor (EGF) and 10(-10) mol L(-1) cholera toxin (CT). After removing coexisting fibroblasts several times, we were able to obtain almost pure basal epidermal keratinocytes. Our technique supports sustained multiplication of mouse basal keratinocytes for more than 100 population doublings, and they retained the capacity to undergo terminal differentiation when given the appropriate stimulus. The epithelial nature of these cultivated cells was demonstrated both by phase-contrast microscopy and by immunostaining with antikeratin antibodies. EGF and CT, which have been reported to accelerate the cellular growth rate, were essential for successful long-term cultivation during multiple passages. Our technique is very simple. It provides a useful and suitable model for investigations of growth, differentiation and skin remodelling in vitro.

Endothelial cell influence on dorsal root ganglion cell formation.

Although endothelial cells are known to produce neutrotrophic factors, endothelial cell influence on growth and survival of ganglion cells has not been documented. For this reason, a long-term culture technique was modified to obtain dorsal root ganglion (DRG) cells. Cells, among them neurons, were released from clusters into the medium for more than four weeks. These cells were grown together with endothelial cells either (1) in close contact as contiguous co-culture, or (2) on porous inserts for non-contiguous co-culture, and, finally, (3) without endothelial cells for ganglion cell culture. Samples from the cultures were stained for the nuclear Ki-67-antigen to detect proliferating cells, and for neurofilaments (NF) to verify the presence of DRG cells with and without mitotic figures. The contiguous co-culture contained three times as many mitotic DRG cells as other culture set ups. Nerve growth factor had no mitotic effect on the different DRG cultures, although it supported the growth of endothelial cells. It is concluded that a subpopulation of DRG cells is easily harvested from long-term DRG cultures. These DRG cells undergo mitosis when in direct contact with endothelial cells.

Advanced technique for long term culture of epithelia in a continuous luminal–basal medium gradient

The majority of epithelia in our organism perform barrier functions on being exposed to different fluids at the luminal and basal sides. To simulate this natural situation under in vitro conditions for biomaterial testing and tissue engineering the epithelia have to withstand mechanical and fluid stress over a prolonged period of time. Leakage, edge damage and pressure differences in the culture system have to be avoided so that the epithelial barrier function is maintained. Besides, the environmental influences on important cell biological features such as, sealing or transport functions, have to remain upregulated and a loss of characteristics by dedifferentiation is prevented. Our aim is to expose embryonic renal collecting duct (CD) epithelia as model tissue for 14 days to fluid gradients and to monitor the development of tissue-specific features. For these experiments, cultured embryonic epithelia are placed in tissue carriers and in gradient containers, where different media are superfused at the luminal and basal sides. Epithelia growing on the tissue carriers act as a physiological barrier during the whole culture period. To avoid mechanical damage of the tissue and to suppress fluid pressure differences between the luminal and basal compartments improved transport of the medium and an elimination of unilaterally accumulated gas bubbles in the gradient container compartments by newly developed gas expander modules is introduced. By the application of these tools the yield of embryonic renal collecting duct epithelia with intact barrier function on a fragile natural support material could be increased significantly as compared to earlier experiments. Epithelia treated with a luminal NaCl load ranging from 3 to 24 mmol/l were analyzed by immunohistochemical methods to determine the degree of differentiation. The tissue showed an upregulation of individual CD cell features as compared to embryonic epithelia in the neonatal kidney.

An improved technique for culture of rice panicles

An improved technique for long-term culture of rice caryopses is necessary for physiological and genetic studies. Panicles of three rice (Oryza sativa) cultivars `Lemont', `Gummo-byeu' and `Hwasung-byeu' were cultured in liquid media with combinations of light versus dark, panicle position, nitrogen level (5-40 mM), and sucrose level (29–351 mM). Grain growth was increased when panicles were positioned horizontally, partially submerged in the media, owing to greater media contact and apoplastic uptake as observed by fluorescent dyes. The optimal media assimilate supply included 175 mM sucrose and 5 mM nitrogen. Grain fill occurred for up to four weeks; grain dry weight reached 80% of that on intact plants, with 50% germination. This technique should allow for future physiological studies with rice or other panicle-bearing species.

Culturing primitive hemopoietic cells. Long-term mouse marrow cultures and the establishment of factor-dependent (FDCP-Mix) hemopoietic cell lines.

The maintenance of normal primitive hemopoietic cell types in culture for long periods can be achieved either by culture of hemopoietic cells in association with bone marrow-derived stromal cells, as in long-term cultures, or by establishing factor-dependent primitive hemopoietic cell lines (FDCP-Mix cell lines). The long-term culture technique and a reproducible method for establishing FDCP-Mix cell lines will be described here (1,2).

Selective generation of CD8+ T-cell clones from the peripheral blood of patients with cutaneous reactions to beta-lactam antibiotics.

The presence, phenotype, and functional characteristics of peripheral blood penicillin-specific T lymphocytes in individuals with cutaneous allergic reactions to penicillin were investigated using in vitro long-term culture techniques. Peripheral blood mononuclear cells from two penicillin-allergic patients were stimulated in vitro with penicillin, and T-cell blasts were clonally expanded by limiting dilution. Seven T-cell clones were derived, all of which were CD3+ CD4- CD8+ HLA-DR+, and produced IL-2 and IFN-gamma upon stimulation. T-cell proliferation required the presence of antigen and autologous, but not allogeneic, antigen-presenting cells. In addition to the parent compound, the T-cell clones also developed a proliferative response to penicilloyl, the major metabolite of penicillin. The cloned T-cell lines were found to exhibit marked suppressor activity for Con A mitogenesis. The observed suppressor activity required cell-to-cell contact, as supernatants from these T-cell clones had no comparable inhibitory effect. These findings indicate that there is a predominance of penicillin-specific CD8+ T cells in the peripheral blood of individuals sensitized to beta-lactam antibiotics.

Erythroid lineage‐specific activity in conditioned medium derived from cloned human marrow stromal cells (CFU‐RF)

Using long-term culture techniques, it has been shown that stromal cells in the marrow microenvironment are essential for the continued production and self-renewal of hematopoietic stem cells. We previously reported the development of a methylcellulose colony assay for a population of marrow stromal progenitors called CFU-RF. In this paper, a method is described for subculturing cells from individual CFU-RF-derived colonies to allow conditioned medium production (StCM). StCM, prepared in this way, was found to possess an erythroid lineage-specific activity that stimulated the formation of macroscopic erythroid colonies in cultures containing erythropoietin (epo). Using dose-response curves, the KG1 colony assay, and antibody neutralization, it was shown that the activity could not be attributed to interleukin 3 (IL3) or granulocyte-macrophage colony-stimulating factor (GM-CSF). However, it was further shown that a monolayer of stromal cells, which had earlier been producing the erythroid activity, could be stimulated by IL1 to produce granulocytic colony-stimulating activity, but only as long as IL1 was present in the culture medium. These findings indicate a mechanism whereby the same stromal population could be modulated to promote growth and differentiation of different hematopoietic lineages.