The Cell Culture Core will be responsible for the establishment, maintenance, and passaging of human keratinocytes from foreskin and adult skin samples. The work required of the Cell Culture Core technicians is more labor-intensive than routine cell culture activities. In addition to routine immersion type cultures, they will prepare lifted cultures, including formulation of matrix and feeder cell coatings, as well as growth and passage of cells lifted to the air/medium interface. The tedious and meticulous care required to prepare lifted and submerged cultures results from the need to coat each Nylaflo membrane in a collagen solution, and transfer it individually to a Petri dish, then covering it with another 1 ml of the collagen solution. Upon gelation, and after a 24 hour rinse with sterile distilled water, cells are plated onto the membranes and grown to confluence (7-10 days). Upon confluence, each membrane has to be carefully removed (lifed with forceps) from the initial dish (avoiding loss of any collagen from the side of the dish) and transferred to a larger dish containing two stacked, glass-wool filters, pre-soaked with medium. Submerged cultures must also be lifted and transferred to new dishes (without filters). Since each experiemtn generally consists of from 40 to 60 dishes, this activity is extremely time-consuming. The Cell Culture Core will prepare delipidized sera by two different techniques. Delipidization and dialysis of serum is time-consuming, requiring precision density-gradient centrifugation in potassium bromide at 50,000 rpms for 36-48 hrs. After centrifugation, the serum is dialyzed, using ten changes of PBS over the next four days, then the serum is filtered through a 0.22 um Millipore filter. The butanol/disopropyl ether method of serum delipidization is also labor- intensive. This procedure requires the addition of known volumes of solvent, repeated vigorous shakings, aliquoting, centrifuging, removal of the organic phase, and repetitation of the entire procedure. The serum is then lyophilized, and resuspended in distilled water to the original volume, after which it is filtered just before each use. The Cell Culture Core will store and classify cell lines, prepare cell suspensions for investigators as needed and perform routine assays, such as DNA and protein determinations. Personnel of the Cell Culture Core will do all of the microdocumentation of cultures, as needed by investigators, including phase microphotography, cell viability quantitation, and staining for low magnification visualization of culture densities.
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