An improved serum-free culture system has been developed for normal human epidermal keratinocytes (HK). Short-term clonal growth and differentiation studies are routinely performed in a defined medium consisting of optimized nutrient medium MCDB 153 supplemented with epidermal growth factor, insulin, hydrocortisone, ethanolamine, and phosphoethanolamine. A small amount of whole bovine pituitary extract (wBPE) is added for initiation of primary cultures, for frozen storage, and for serial culture. The need for feeder layers, conditioned medium, serum, and specialized culture surfaces has been eliminated entirely. With an optimal level of calcium ion (0.3 mM), colony-forming efficiency is about 30 percent and cellular multiplication rate is 0.96 doublings per day in the defined medium. A high-calcium concentration (1.0 mM) induces stratification and terminal differentiation, which can be quantified by counting cornified envelopes that are resistant to boiling in sodium dodecyl sulfate plus dithiothreitol. Under optimal conditions with wBPE present, cellular senescence occurs after about 40 population doublings. Scanning electron microscopy (SEM) has verified the occurrence of stratification during differentiation in the defined medium with high calcium. High-voltage electron microscopy (HVEM) after detergent extraction of human epidermal keratinocyte (HK) colonies grown in the defined medium with low and high calcium has revealed specific changes in the intermediate filament network and keratohyalin granules corresponding to changes in cellular differentiation. Indirect immunofluorescence studies have verified that the intermediate filament network observed with HVEM is composed of keratin proteins.
A serum-free medium with bovine pituitary extract as the only undefined supplement has been developed for long-term culture of human mammary epithelial cells. This medium supports serial subculture of normal cells for 10-20 passages (1:10 splits) without conditioning or special substrates, and it supports rapid clonal growth with plating efficiencies up to 35%. It consists of an optimized basal nutrient medium, MCDB 170, supplemented with insulin, hydrocortisone, epidermal growth factor, ethanolamine, phosphoethanolamine, and bovine pituitary extract. Replacement of pituitary extract with prostaglandin E1 and ovine prolactin yields a defined medium that supports rapid clonal growth and serial subculture for three or four passages. Cultures initiated in these media from normal reduction mammoplasty tissue remain diploid and maintain normal epithelial morphology, distributiop of cell-associated fibronectin, expression of keratin fibrils, and a low level of expression of milk fat globule antigen. Large cell populations can now be generated and stored frozen, permitting multiple experiments over a period of time with cells from a single donor. These media greatly extend the range of experiments that can be performed both conveniently and reproducibly with cultured normal and tumor-derived human mammary epithelial cells.Growth of human mammary epithelial cells (HMEC) in medium MM, which contains hormones, fetal bovine serum, and conditioning factors from human epithelial and myoepithelial cells, has been reported previously (1,2). This medium supports rapid monolayer growth of normal HMEC for three or four passages with 1:10 splits. However, good clonal growth in MM requires cholera toxin and a fibroblast feeder layer (3). Tumor-derived cells respond similarly, but their doubling potential is more variable.Although medium MM makes possible many types of experiments with cultured HMEC (4-7), its usefulness is limited by lack of definition and by early cellular senescence. Other culture systems described for HMEC are generally even more restrictive (reviewed in ref. 8).This report describes long-term growth of HMEC with pituitary extract as the only undefined supplement and shorter-term monolayer and clonal growth in a defined medium. These results were achieved by applying to HMEC methods previously used in similar studies with other cell types (9-11). MATERIALS AND METHODSMedia and Solutions. Medium MM was prepared as previously described (2), except that HS578 Bst conditioned medium and cholera toxin were omitted. Medium MCDB 170 (Table 1) is an optimized formulation for HMEC. Medium MCDB 202a differs from MCDB 170 in concentrations of four components: cysteine HCl, 2.0 x 10-4 M; glutamine, 1.0 X 10-3 M, sodium pyruvate, 5.0 x 10-4 M, and zinc sulfate, 1.0 x 10-7 M. MCDB 202a is a minor modification of previously published medium MCDB 202 (13). MCDB 170 and MCDB 202a were both prepared according to the protocol for MCDB 104 (12), with appropriate adjustment of concentrations. These media were prepared withou...
Colony formation by normal human epidermal keratinocytes (HK) has been achieved in a medium that contains no deliberately added undefined supplements. The term "defined" is used to describe this medium, although the possibility that trace contaminants in its components could be contributing to the multiplication that it supports cannot yet be ruled out completely. The defined medium consists of a basal medium, MCDB 152, supplemented with 5 ng/ml epidermal growth factor (EGF), 10 micrograms/ml transferrin, 5 micrograms/ml insulin, 1.4 X 10(-6) M hydrocortisone, 1.0 X 10(-5) Methanolamine, 1.0 X 10(-5) M phosphoethanolamine, and 2.0 X 10(-9) M progesterone. MCDB 152 differs from MCDB 151, previously developed for multiplication of HK with small amounts of dialyzed serum (Peehl and Ham, 1980b), only by addition of the trace element mixture from human fibroblast medium MCDB 104 (McKeehan et al., 1977). Most of the requirement for transferrin, which is the least defined component of the defined medium, can be replaced by adding freshly dissolved and sterilized ferrous sulfate to the final medium after it has been filter sterilized. Insulin and EGF are clearly needed for optimal multiplication and hydrocortisone is mildly beneficial. Either ethanolamine or phosphoethanolamine must be present in the defined medium for HK multiplication. There is a greater need for EGF and less for hydrocortisone in the defined medium than in previous partially defined systems that we have worked with. Very large colonies of flattened epithelial cells are obtained in the defined medium, which has a low calcium concentration (0.03 mM) and does not favor keratinocyte differentiation. Less growth and more differentiation are obtained with higher calcium concentrations. The defined medium is highly selective for keratinocyte growth from a mixed inoculum of keratinocytes and fibroblasts.
Clonal growth of cultured mammalian cells with high plating efficiency in unrestricted volumes of medium was first reported by Puck, Marcus, and Cieciura,' and has been subsequently verified in many other laboratories. However, prior to the present studies, such growth has always required the presence in the medium of whole or dialyzed serum, purified serum proteins,2 or unknown factors dialyzed into the medium from serum.3 In the current series of investigations, the nutritional requirements for single cell plating of near-diploid Chinese hamster cell lines have been studied in detail,4-7 and conditions have now been achieved which routinely permit rapid clonal growth in a completely synthetic medium. Materials and Methods.-Cell lines: Strain CHD-3A (Puck) is a subclone derived from an essentially diploid Chinese hamster ovary line isolated in 1957.8 CHD-3A is a subdiploid strain, with most of its cells containing 21 chromosomes. It and its immediate precursors were used in the previous studies of this series.4-7 Strain CHL-1 (Puck) is a nonclonal line derived from the lung of a newborn male Chinese hamster.5 Current cultures of strain CHL-1 show a preponderance of cells with 23 chromosomes. Mouse L cells, strain 929 (Earle), were obtained from Dr. Donald King.Stock cultures: Stock cultures of all three lines were maintained as monolayers on glass in medium designated FlOPFC, which consists of nutrient mixture F10,5 supplemented with 0.10 mg/ml "panmede" (Paines and Byrne, Ltd., Greenford, England) and 2% fetal calf serum. All experiments described in this paper were initiated with cells previously grown in medium F1OPFC.Culture vessels: Plastic tissue culture Petri dishes (Falcon Plastics Co. no. 3002) were used for all plating and serial culture experiments. Glass surfaces, prepared by the procedures normally used in this laboratory for single cell plating, support, at best, only sparse growth in synthetic
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.