Dendritic Cell Culture

Ocular drugs, cosmetics or all chemicals, which can come in contact with the eye carry the potential of harming various ocular structures. Thus, their possible toxic effects on ocular structures must be evaluated in tests before use in humans. 

Introduced by Wolfgang H. Vogel, Jefferson Medical College, Thomas Jefferson University, Department of Pharmacology, Philadelphia, USA 

A comprehensive review of various in vitro and in vivo procedures has been published recently by Wilhel-mus in 2001. 

Dart J (2003) Corneal toxicity the epithelium and stroma in iatrogenic and factitious disease. Eye 17 886-892 Wilhelmus KD (2001) The Draize Test. Surv Ophthalmol 45 493-515 

This test uses an in vitro human dendritic cell culture to obtain information of the potential for various chemicals to induce allergic contact dermatitis. This test is used as an alternative to the Local Lymph node assay (LLNA) to minimize or replace the use of live animal testing for predicting skin sensitization (Kimber et al. 2002, see below). The test allows for evaluation of skin sensitization by examining the presence of cell surface markers on Periperal Blood Mononuclear Cell (PBMC)-derived dendritic cells (DC) that are known to be involved in the development of allergic contact dermatitis. 

---

Hymery N, Sibiril Y, Parent-Massin D (2006) Improvement of human dendritic cell culture for immunotoxicological investigations. Cell Biol Toxicol 22 243-255... 

Experiments have also been performed with primary macrophage and dendritic cell cultures (64). However, these have only been performed when it is necessary to show that the virus will only grow in macrophages and not in T cells. Since macrophage-tropic HIVs grow well in PHA blasts, and culturing... 

Upregulation of ILIO and IL12p40 secretion was observed in human monocyte-derived immature dendritic cells cultured with an aqueous extract from Iceland moss (Freysdottir et al. 2008). 

Inaba, K. et al., Generation of large numbers of dendritic cells from mouse bone marrow cultures supplemented with granulocyte/macrophage colony-stimulating factor, J. Exp. Med., 176, 1693, 1992. 

IL-23 has recently been shown to be produced by dendritic cells and by human cultured keratinocytes in healthy skin and in psoriasis - its role in atopic dermatitis has to be defined [22]. Interaction of keratinocytes with activated T cells via CD40-CD40L may enhance IL-23 production and subsequently the IFN-y production by memory T cells [23]. 

CD25 + T-regulatory cells can markedly inhibit allogeneic dendritic cell-stimulated MLR cultures. Blood 2004 104 453-461. 

To characterize IRIV-elicited immune responses in vitro, we addressed cell proliferation and cytokine expression in peripheral blood mononuclear cell (PBMC) cultures, as well as IRIV effects on dendritic cells (DC). In all experiments, PBMC were obtained from healthy donors and, if needed, further separated into different cell subsets. Finally, cells were cultured in the presence or absence of IRIV as indicated. 

First, we incubated immature dendritic cells (iDC) in the presence or absence of IRIV and could not observe upregulation of defined maturation markers. However, iDC incubation with supernatants harvested from IRIV stimulated PBMC cultures resulted in upregulation of CD86, human leukocyte antigen (HLA)-class I molecules, and, in most cases, also of CD83 (6). These results demonstrate that IRIV-induced cytokine secretion of PBMC indeed favors maturation of DC. 

Figure 6 Immunopotentiating reconstituted influenza virosomes (IRIV) adjuvant effects in the induction of tumor associated antigen-specific cytotoxic T cell. CD14-negative cells from a healthy donor peripheral blood mononuclear cells were cocultured with autologous immature dendritic cells (iDC) in the presence of Melan-A/Mart-l27-35, alone (A) or supplemented with either control liposomes (B) or IRIV (1 50, C). On day 7, culture cells were restimulated with Melan-A/MART-127-35 pulsed iDC and cultured for six further days [see Materials and Methods ]. On day 7 after restimulation cells were stained with fluorescein isothiocyanate-conjugated anti-CD8 and phosphatidylethanolamine-conjugated HL A-A0201 /Melan-A/MART -127-3 5 tetramers. Source From Ref. 6.

COS-7 or CHO cells (for initial transfection screening) or cells of therapeutic interest (e.g., dendritic cells and various cancer cells) at a confluence of 50%, grown in 96-well culture plates, were placed into the robot by the robotic conveyor. In a fully automated process, the robot removes the lid from the cell culture microtiter plate, dispenses lipoplexes into the wells (triplicates), replaces the lid and returns the plate to the incubator. After four hours, the cells are automatically retrieved, the cell monolayers are carefully washed using a special drop mode of the integrated plate washer, fresh medium is added, and the cells are incubated for further 42 hours before harvesting. 

Estrogens stimulate sprouting of axons and dendrites in neurons in cell cultures, and there is preliminary evidence that the hormone improves memory in healthy women and in those with Alzheimer disease.285... 

Lou E, Marshall J, Aklilu M, Cole D, et al. 2006. A phase II study of active immunotherapy with PANVAC or autologous, cultured dendritic cells infected with PANVAC after complete resection of hepatic metastasis of colorectal carcinoma. Clin Colorectal Cancer 5 ... 

Kiertscher SM, Roth MD. Human CD14+ leukocytes acquire the phenotype and function of antigen-presenting dendritic cells when cultured in GM-CSF and IL-4. Journal of Leukocyte Biology 1996, 59, 208-218. 

Bodewes, R., Geelhoed-Mieras, M.M., Heldens, J.G.M., Glover, J., Lambrecht, B.N., Fouchier, R.A.M., Osterhaus, A.D.M.E., Rimmelzwaan, G.F. The novel adjuvant CoVaccineHT increases the immunogenicity of cell-culture derived influenza A/H5N1 vaccine and induces the maturation of murine and human dendritic cells in vitro. Vaccine 27 (2009) 6833-6839. 

---