Trypsin-containing cell suspension

Techniques are also available for the separation of various cell types in the central nervous system. Among the most widely used methods to separate neuroglial cells from neurons are the techniques described by Sellinger and Azcurra (1974), but other techniques using acetone-glycerol separation of cells or trypsinization of cell suspensions are also available (see Rose, 1968, for a discussion). Methods for the isolation of myelin, postsynaptic membranes, lysosomes, and the vesicles present within nerve terminals that contain neurotransmitters are also available. Invariably, these preparative methods employ some form of density-gradient centrifugation. 

Calf kidneys, dog kidneys and rhesus monkey kidneys were treated with trypsin to give suspensions of cells. The suspensions were centrifuged and the packed cells diluted with 400 volumes (calf cells) or 200 volumes (dog cells and rhesus monkey cells) of a growth medium consisting of 5% horse serum and 0.5% lactalbumen hydrolysate in Earle s saline, with 100 units/ml each of penicillin and streptomycin. These media were used separately to produce Semliki Forest/calf interferon, Semliki Forest/dog interferon and Semliki Forest/rhesus monkey interferon. The cell-containing growth medium was dispensed into 500 ml medical flat bottles (70 ml in each). The cultures were incubated at 36°C. Confluent sheets of cells (monolayers) were formed in 5 to 6 days. The growth medium was then removed and the monolayers were washed with isotonic phosphate-buffered saline, pH 7.5. 

Animal cell cultures that are initiated from cells removed directly from the animal are called primary cultures (Figure 2). Primary cultures include both explant cultures (i.e., cultures initiated from small pieces of intact tissue), as well as cultures initiated from preparations of individual or dispersed cells (obtained from intact tissue by mechanical or proteolytic dismption). Nerve fiber explant cultures in blood plasma were among the earliest types of tissue cultures (Harrison, 1907). Cells grow out from such tissue explants and form a single layer of cells completely filling the tissue culture vessel surface. Such cell cultures are called confluent monolayers. Confluent monolayers can then be treated with trypsin, so as to remove the individual cells from the culture vessel surface. The resulting cell suspension is then transferred into other culture containers, so that more viable monolayer... 

The trypsinized cell suspension is counted and diluted in complete media before assessing for survival. For each treatment set up five Petri dishes containing 200 cells per dish. 

Fibroblast cultures for each patient and control are grown to confluency in T-75 culture flasks. The assay is then performed in triplicate using six-well plates. First, 1 ml of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA) solution (Sigma T4049) is added to each T-75 flask. After the cells have lifted from the flask, they are resuspended in 6 ml complete MEM. A 1-ml aliquot of the cell suspension is transferred to a 50-ml conical tube containing 14 ml of MEM complete medium (see above). Following further resuspension, 4 mL is plated into each of three wells in a six-well culture plate and placed into an incubator. Once the cells have attached to the seeded plates and are approximately 90-100% confluent (3-4 days), complete MEM is replaced with 1.5 ml of complete IVPM (see above). 

Trypsinization is performed as described above. Cells in the suspension are counted and prepared in a suspension containing 4.2-6.7 x 104 cells/cm2. To each apical well of the 24-well plate 400 til of the cell suspension are added apically. The Feeder Tray contains 40 ml of complete medium (feeding cells from the basolateral side). 

Cell and tissue implants Cell suspensions are obtained by trypsinization of confluent cell monolayers. Five microliters containing 2 X 10 cells in medium supplemented with 10% serum are introduced in the corneal micropocket. When the over expression of growth factors by stable transfection of a specific cDNA is studied, one eye is implanted with transfected cells and the other with the wild type cell line. A 0ien tissue samples are tested, samples of 2-3 mg are obtained by cutting the original fragments under sterile conditions. The angiogenic activity of tumor samples is compared with macroscopically healthy tissue. 

Connect four containers to the multiplate-end connection aseptically. The first vessel contains the trypsin solution and the second contains the growth medium required to dilute the cells and inactivate the trypsin. The third and fourth vessels are used to capture the spent medium (supernatant) and the cell suspension, respectively. The recommended procedure for connecting the vessels to the multiplate-end connection is described in detail in Steps 2-4. 

Once cells are 90 % confluent, remove the growth medium and wash the cells 2x with FIBSS for eliminating floating cells and debris. Trypsinize the cells and transfer the suspension to 15 mL tube containing 1 mL DMEM/FBSplus 1 %penicillin/streptomycin. Wash the culture dish with 3 mL DMEM/FBS plus 1 % penicillin/ streptomycin to collect any remainder cell and transfer it to the same tube. Spin the tube at 269 for 10 min at 4 °C. Carefully remove the supernatant, add 2 mL DMEM/FBS plus 1 % penicillin/ streptomycin, and resuspend the cells using a P200. Complete to a 5 mL final volume DMEM/FBS plus 1 % penicillin/streptomycin. Spin the tube at 269 x i for 5 min at 4 °C. 

Cells from human or other primate sources are obtained from an intact tissue, e.g. human embryo kidney or monkey kidney. The cells are dispersed by digestion with trypsin and the resulting suspension of single cells is generally allowed to settle in a vessel containing a nutrient medium. The cells will metabolize and grow and after a few days of incubation at... 

Collect suspension cell lines or cells detached from the dishes by trypsinization in a 15-ml conical tube and wash with BSS containing 2 mM CaCla three times. 

Soluble recombinant protein extracted fiom cells or supernatants of eukaryotic cells (e.g, Chinese hamster ovary cells or insea cells expressing recombinant baculovlrus) or baaeria (such as . colt harbouring plasmids or recombinant viruses) and dissolved in PBS Protein separated electrophoretically in sodium dodecyl sulfate-containing polyacrylamide gels (SDS-PAGE) and eluted from gel slices into PBS Cultured cells grown in suspension or as monolayers in flasks, then detached by treatment with 0,1% trypsin-EDTA, and resuspended in PBS or serum-free DMEM... 

---