Tissue differentiation, culture media

Over 300 peptides isolated in our laboratory were studied in one or more tumor or normal cell cultures [39-44]. Part of the results obtained is summarized in Table 2.3. Over 75% of the peptides showed pronounced proliferative or antiproliferative activity in at least one cell type (Fig. 2.3). As a rule, tumor cells are more sensitive to peptide action. Besides the cell type, experimental conditions such as cell density or composition of the culture medium also affected the overall effect. In several cases (13%, Fig. 2.3) even the sign of the effect was peptide concentration dependent. Generally, experiments with cell cultures conform with the view that the main physiological function of cell and tissue peptidomes is control of long term processes and the homeostatic balance (i.e. cell differentiation, proliferation and elimination). The overall effect of peptide pools is achieved by concerted action of total sets of peptides rather than by single components. The molecular mechanisms of peptide action in cells requires concrete study in each individual case and are the subject of current research. 

Cells freshly extracted and isolated from an organ or tissue and plated in a defined culture medium (e.g., PBS or L-15 media). During this procedure two parallel processes occur 1) differentiated cells of the original tissue explants usually do not divide and, with time, will successively lose some of their specialized functions (dedifferentiation) and 2) decrease of number of specialized cells (e.g., fibroblasts divide rapidly, and will eventually outnumber the specialized cells). Volume 1(14). 

Note the monocytes will differentiate into adherent macrophages by day 7 of incubation. To harvest cells, discard the culture medium and wash once with 25 mL of cold PBS without Ca++ or Mg++. Add 20 mL of fresh cold PBS to the flask and incubate on ice until cells begin to round-up and detach (usually 15-20 min) (see Note 14). Gently scrape cells into the PBS with a cell scraper and transfer the cell suspension to a 50-mL conical centrifuge tube. Pellet the cells by centrifugation at 500 x g for 5 min. Resuspend cells in 5-10 mL of RPMI plus 10% FBS and 50 ng/mL M-CSF. Count cells, add medium to adjust to the desired cell concentration, and re-plate in a tissue culture plate (see Note 15). Incubate at 37 °C in 5% CO2 for at least 6 h to allow macrophages to adhere. 

Fig. 2A-C. Diagrammatic representation of stem or leaf inoculation with Agrobacterium rhizogenes to produce transformed roots, A Surface-sterilized stem or leaf explants are placed on a sterile flat surface and gently wounded with a hypodermic needle. Two to three drops of A. rhizogenes suspension ( 2 x 10 bacteria) are applied to the wound spot. B Tissues are cultured in nutrient agar (MS or B5 medium + 3% sucrose) such that the wounded portion does not come in contact with the medium. C After about 2 weeks at 20-25 °C, roots usually differentiate around the wound spot. These wound spots are cultured in 20-50ml liquid medium, with 500 ig/ml cefotaxime or ampicillin to kill the bacteria...

Skin cells from the body do not differentiate when ttiey are simply placed in a tissue culture medium that is, they do not organize into the structure of skin, with different layers and different cell types. What is needed to cause such differentiation to occur Indicate the most important requirements on any material used. 

Several authors have studied nicotine production (i.e., biosynthesis) in callus tissue cultures (Speake et al., 1964 Benveniste et al., 1966 Furuya et al.y 1966, 1971 Tabata et aL, 1968, 1971 Shiio and Ohta, 1973 and Heinze, 1975). The biosynthesis of nicotine is dependent upon the formation of organized tissue within the callus. Nodule-like structures similar to roots were observed in our laboratories using tobacco variety Maryland-872, which produces 96% of its alkaloids as nicotine. Shoot formation stimulated nicotine production in the callus, and nicotine may have been transported from the callus to the shoot. Nicotine production and tissue differentiation were dependent upon concentrations and types of growth regulators in the culture medium (Tables 4.3 and 4.4). The vegetative buds and leaves (shoots) contained about live times as much nicotine as callus without buds or leaves, which is in agreement with the results of Tabata et al (1968). 

The rate of mesenchymal stem cell (MSC) proliferation in monolayer environment is slow, a problem which needs to be addressed in view of the need to obtain sufficient number of cells within a reasonable time frame./n vitro growth and activities of MSCs are supported and enhanced by biochemical such as cell culture medium. These include-growth factors, cytokines, and hormones.However,it is recently reported that mechanical forces also play acentral role in the physiological process involving a wide variety of tissues [6-9]. Cyclic uniaxial strain applied on elastic substrates causes changes in cell responses, orientation and alignment.The application of mechanical force also influences cell proliferation, differentiation, and gene expression in a wide variety of cell types [10-12]. 

The manner in which this transition from cell division to differentiation is regulated is largely unknown. We shall discuss a few relevant findings later (page 231). If this regulatory mechanism is disturbed, then cell division can occur without subsequent differentiation. The disturbances can be of quite different kinds. For example, one can take tissue from the whole plant and grow it in a defined culture medium. Under certain conditions, such tissue cultures can maintain their ability to divide almost indefinitely (page 246). 

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