Frizell L. Vaughan

Keratinocytes grown at the air-liquid interface

SummaryA procedure is described which allows primary cultures of rat keratinocytes grown at the liquid-air interface to develop and maintain multilayered strata and to produce highly keratinized sheets morphologically similar to those seen in epidermis in situ. Various substrata were tested and compared as to their ability to support growth and stratification of keratinocytes. It was found that when cultured on plastic surfaces, keratinocytes adhered tightly to the substratum and produced a confluent monolayer that later stratified to two to three layers. Cells plated on Vitrogen 100 collagen failed to reach confluence and, in addition, exhibited the “clustering” phenomenon and deterioration of collagen after 3 to 4 d of growth. Significantly better attachment and spreading were observed for cells grown on rat-tail collagen as compared with plastic and Vitrogen 100 collagen. The best results, including maximal and uniform stratification, were seen in cells grown on a mixture, of rat-tail and Vitrogen 100 collagens.The system that was developed in the present study offers a model for use in the study of epidermal toxicity from topically applied environmental chemicals.

Growth and differentiation of primary rat kerationocytes on synthetic membranes

SummaryThe attachment, proliferation, and differentiation of primary cultures of kerationcytes isolated from murine epidermis were monitored after purified cell suspensions were seeded and incubated in vitro on various synthetic membranes. Concomitant studies of the effects of attachment factors added to synthetic membranes before use as substrata for keratinocytes were also done. The study demonstrated that a synthetic membrane composed of nylon was superior to other membranes and to plastic control culture vessels in supporting the growth of murine keratinocytes. Although laminin enhanced initial attachment and proliferation of cells on nylon membranes, the untreated substratum was more effective for extended incubation. Stratification and differentiation of these kerationcytes on the nylon substratum was enhanced by raising confluent cultures (7 d) to the air-medium interface so that they were in contact with medium only from the bottom. Cultures raised for 14 d produced many morphologic markers of the epidermis and closely resembled the architecture of this tissue in situ.

Requirement of hydrocortisone and insulin for extended proliferation and passage of rat keratinocytes

SummaryA procedure for the preparation and cultivation of rat epidermal basal cells from full thickness skin resulted in greater than 99% viability and 90% plating efficiency. However, attempts to subculture monolayers of these epithelial cells grown in medium with serum as the only supplement were totally unsuccessful. When hydrocortisone and insulin were added to the medium, subcultivation of primary growth was obtained. It was demonstrated that hydrocortisone at concentrations as low as 0.1 μg/ml was necessary for at least the initial attachment of the cells to the substrate—an essential step in subcultivation. Increasing concentrations of insulin (0.1 to 50 μg/ml) caused the rate of proliferation and the cell density to increase, but insulin alone did not support subcultivation.

Stratified cornified primary cultures of human keratinocytes grown on microporous membranes at the air-liquid interface

It was previously reported that rat keratinocytes grown at the air-liquid interface on collagen gels or on nylon membranes produce multilayered cultures of uniformly stratified cells, comparable to the epidermis in situ by morphological and biochemical criteria. A protocol has now been developed by which primary human keratinocytes grown for two weeks submerged on microporous nylon membranes and raised to the air-liquid interface for an additional three weeks, exhibit most of the comparable characteristics of the epidermal cells in vivo. Staining with fluorescein isothiocyanate-conjugated monoclonal antibodies indicated the presence of 56,5 and 65-67 kDa keratins as well as filaggrin-type proteins in the upper cellular layers. Desmosomes, lamellar granules and keratohyalin-like granules were observed. Cultures were covered with layers of cornified cells. This study differs from the majority of other investigations on human keratinocytes in that no feeder layers or other biological substrata were used. This system should be useful in toxicological studies of chemicals which are to be applied topically to the skin.

Molecular aspects of control in epidermal differentiation

SummaryThe mammalian epidermis is organized into layers of structurally different cells—the basal, spinous, granular and cornified layers—which represent steps in the differentiative process that terminates in cornification and desquamation. Investigation of the molecular mechanisms that control this ordered sequence of events provides clues to the etiology of certain epidermal pathologies. DNA synthesis and mitosis are normally restricted to the basal layer. Several substances have been implicated in the mitotic control of epidermal cells, the loss of mitotic activity being the first major step in normal keratinization. Investigations performed in this laboratory indicate that isolated differentiated nuclei can replicate their DNA which they are inhibited from doingin situ. Addition of a high speed supernate from homogenized differentiated cells inhibited this synthetic activityin vitro suggesting the existence of a cytoplasmic inhibitor of DNA synthesis. It is not known whether mitotic inhibition in differentiated epidermal cells is a function of the inhibition of DNA replication.Contrary to previous assumptions, recent experimental evidence clearly indicates that, unlike DNA synthesis, RNA synthesis occurs in differentiated cells. Correlated with this synthetic activity is the observation that a protein rich in histidine is specifically formed in the granular cells. This protein appears to be a component of the keratohyalin granules which fill the cells of the granular layer. Investigations were conducted in this laboratory to determine whether control of the synthesis of this protein occurs at the level of translation or transcription. Translation,in vitro, of mRNA obtained from isolated populations of each epidermal cell type suggested that control of protein synthesis in the differentiating epidermis is transcriptional, i.e. only in the granular cell is there an mRNA for the histidine-rich protein. Transcription,in vitro, of chormatin isolated from the separated cell populations produced RNA with a ratio of cytidine to uracil consistent with the predicted mRNA for this protein thus providing additional support for the hypothesis that epidermal differentiation is controlled at the level of ‘gene-readout.’

Bis(2-chloroethyl)sulfide (BCES) disturbs the progression of rat keratinocytes through the cell cycle

Epidermal basal keratinocytes are the primary target in BCES-induced cutaneous injury. DNA synthesis is inhibited by exposure to BCES which could relate to the mustards cytotoxic effect. The effects of BCES on the cell cycle in keratinocytes synchronized by aphidicolin were investigated. Primary keratinocytes synchronized at the G1/S boundary entered the S, G2, M, and G1 phases at successive times after release from the block. When cells were exposed to 1, 10, or 50 microM BCES in different phases of the cell cycle, cells in the S phase were more sensitive to BCES than cells in the other phases. Keratinocytes exposed to 1 microM BCES at the G1/S boundary exhibited a prolongation of the S phase and a block in the G2 phase. When these cells were exposed to 10 or 50 microM BCES, they did not enter the S phase for up to 12h and the incorporation of thymidine into DNA was inhibited. These results suggest that the blocks in the G2 and G1 phases relate to the cytotoxic effect of BCES on the germinative population of epidermal keratinocytes.

Failure to observe a relationship between bis‐(β‐chloroethyl)sulfide‐induced NAD depletion and cytotoxicity in the rat keratinocyte culture

It has been proposed that the activation of poly(ADP-ribose) polymerase (Papirmeister et al., 1985), which results from the presence of strand breaks in bis-(beta-chloroethyl)sulfide (BCES) damaged DNA, causes depletion in the level of nicotinamide adenine dinucleotide (NAD) leading to cell death. This hypothesis has now been evaluated in the primary submerged culture of rat keratinocytes. The DNA content, the viable cell number, and the proliferative capability (measured by thymidine incorporation) of the culture were all reduced 48 h after exposure to 10 microM BCES. However, the total NAD level, that is, NAD+ plus NADH, was not changed at a dose of BCES lower than 50 microM. This observation was the same in both proliferating and early differentiating cultures. To further test this hypothesis, the modifying effect of inhibiting poly(ADP-ribose) polymerase on cytotoxicity in BCES-exposed cells was investigated. After exposure to 250 microM BCES, the NAD level was reduced to approximately 26 pmol/micrograms DNA. This value was increased to 34-49 pmol/micrograms DNA at both 24 and 48 h postexposure when the cultures were incubated in medium supplemented with 1-10 mM nicotinamide. Nevertheless, the decrease in the DNA content of the culture was not reversed. These results suggest that in the rat keratinocyte culture exposed to BCES, depletion of NAD is not a prerequisite for cell death.

Pseudoepidermis, constructed in vitro, for use in toxicological and pharmacological studies.

The purpose of this study was to establish the validity of the stratified, cornified keratinocyte culture as a model for investigating cutaneous toxicities. This pseudoepidermis, grown on a nylon membrane at the air-liquid interface, responded to topical application of a known vesicant similarly to the response of the tissue in vivo. Alterations in the morphology of the in vitro model also resembled pathological changes seen in in vivo models after exposure to this agent. The effects of the skin irritants benzoate and salicylate on protein and DNA synthesis in the culture were also similar to those observed in vivo.

Isolation, Purification, and Cultivation of Murine and Human Keratinocytes

The architecture of mammalian skin incorporates an outer layer of stratified epithelium. This enables the organism to conserve internal homeostasis and maintain protection from adverse environmental exposure. The keratinocyte is the cell primarily responsible for this structure. Isolation and in vitro cultivation of this cell type is widely used in dermatological and other investigations as opposed to using whole animals. However, this cell is very fastidious as compared to other skin cells (fibroblasts, etc.) and thus requires special procedures to obtain successful in vitro cultivation. This chapter describes the methodology required to isolate, purify, and cultivate keratinocytes to produce both monolayer and stratified cultures. The methodologies for producing cultures of keratinocytes obtained from rat skin and from human skin are described.