Keratinocyte Serum-free Medium Research Articles

Uroepithelial cells can directly respond to Mycobacterium bovis bacillus Calmette‐Guérin through Toll‐like receptor signalling

To investigate, in a human urinary tract cell line, the interaction of Toll-like receptor (TLR) signals with cytoplasmic adapter proteins MyD88 and bacillus Calmette-Guérin (BCG), and evaluate the epithelial cytokine response to BCG infection. Intravesical BCG therapy is effective against carcinoma in situ and as prophylaxis for recurrence, but although immunological mechanisms have been assumed, the mechanisms of the antitumour effects of BCG have not been completely elucidated. The cell line was first screened for TLR expression by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was isolated from a human urinary cell line, Hu35E6E7, and cDNA synthesised. PCR was used to measure the gene expression of TLR-2, -3, -4, -5, -9, MyD88, MD-2, CD14 and interleukin-8 and -6. The Hu35E6E7 cell line was cultured in keratinocyte serum-free medium, and BCG was added to the cell culture. After Hu35E6E7 cells were stimulated by BCG for various periods, the total RNA of the cells was extracted. Quantitative real-time PCR was conducted for MyD88 using appropriate probes, and the expression of MyD88 analysed. The cell supernatant was collected, and the levels of interferon-gamma, tumour necrosis factor-alpha, interleukin-2, -12, -4, -6, -10, -8 and -1beta were assayed using an enzyme-linked immunosorbent assay. Uroepithelial cells expressed TLR-2, -3, -4 and -9, and MyD88, MD2, CD14, interleukin-6 and -8 were also detected. At 3, 6, 9 and 12 h after adding BCG, quantitative PCR assay showed that the expression of MyD88 was maximal at 6 h. The presence of BCG stimulated the release only of interleukin-6 and -8 from Hu35E6E7 cells after 6 h. By contrast, interferon-gamma, tumour necrosis factor-alpha, interleukin-2, -12, -4, -10 and -1beta were not detected in the culture supernatant. These results show that uroepithelial cells, but not immune cells, responded directly to BCG through TLR signalling. Further investigation is needed to determine the role of cytokines released from uroepithelial cells after BCG infection.

Enhancement of Streptolysin O Activity and Intrinsic Cytotoxic Effects of the Group A Streptococcal Toxin, NAD-Glycohydrolase

Streptolysin O (SLO) is a cholesterol-dependent cytolysin produced by the important human pathogen, group A Streptococcus (Streptococcus pyogenes or GAS). In addition to its cytolytic activity, SLO mediates the translocation of GAS NAD-glycohydrolase (NADase) into human epithelial cells in vitro. Production of both NADase and SLO is associated with augmented host cell injury beyond that produced by SLO alone, but the mechanism of enhanced cytotoxicity is not known. We have now shown that expression of NADase together with SLO dramatically enhanced the lytic activity of GAS culture supernatants for erythrocytes but had no effect on SLO-mediated poration of synthetic cholesterol-rich liposomes. This result revealed a previously unknown contribution of NADase to the cytolytic activity associated with GAS production of SLO. Purified recombinant SLO bound NADase in vitro, supporting a specific, physical interaction of the two proteins. Exposure of human keratinocytes to wild-type GAS, but not to a NADase-deficient mutant strain, resulted in profound depletion of cellular NAD+ and ATP. Furthermore, expression of recombinant GAS NADase in yeast, in the absence of SLO, induced growth arrest, depletion of NAD+ and ATP, and cell death. These findings have provided evidence that the augmentation of SLO-mediated cytotoxicity by NADase is a consequence of depletion of host cell energy stores through the enzymatic action of NADase. Together, the results have provided mechanistic insight into the cytotoxic effects of a unique bipartite bacterial toxin.

Adherence and blocking of Candida albicans to cultured vaginal epithelial cells: treatments to decrease adherence.

Background. Pathogenesis of mucosal microorganisms depends on adherence to the tissues they colonize and infect. For Candida albicans, cell surface hydrophobicity may play a significant role in tissue binding ability. Methods. A continuous cell line of vaginal epithelial cells (VEC) was grown in keratinocyte serum-free medium (KSFM) with supplements and harvested by trypsinization. VEC were combined with yeast cells to evaluate adherence and inhibition of adherence. In this experimental setup, yeast stained with fluorescein isothiocyanate were allowed to attach to VEC and the resulting fluorescent VEC were detected by flow cytometry. Results. VEC were cultured and examined daily after plating and showed morphology similar to basal epithelial cells. Culture media supplemented with estradiol showed increased VEC proliferation initially (first 24 h) but cell morphology was not altered. Fluorescinated Candida cells bound effectively to the cultured VEC. Using fresh cells exposed to various preparations of K-Y, we showed that all formulations of the product reduced Candida binding to VEC by 25% to 50%. While VEC were generally harvested for use in experiments when they were near confluent growth, we allowed some cultures to grow beyond that point and discovered that cells allowed to become overgrown or stressed appeared to bind yeast cells more effectively. Conclusion. Flow cytometry is a useful method for evaluating binding of stained yeast cells to cultured VEC and has demonstrated that commercially available products have the ability to interfere with the process of yeast adherence to epithelial cells.

Acquisition of Androgen Independence by Human Prostate Epithelial Cells during Arsenic-Induced Malignant Transformation

Lethal phenotypes of human prostate cancer are characterized by progression to androgen independence, although the mechanisms behind this progression remain unclear. Arsenic is a potential human prostate carcinogen that may affect tumor progression. In this study, we used a prostate cancer cell model in which an immortalized, nontumorigenic human prostate epithelial cell line (RWPE-1) had been malignantly transformed by chronic low-level arsenic to help determine whether arsenic affects prostate tumor progression. Control and CAsE-PE (chronic-arsenic–exposed human prostate epithelial) cells were continuously maintained in a complete medium [keratinocyte serum-free medium (K-SFM) with bovine pituitary extract and epidermal growth factor] or in a steroid-depleted medium (K-SFM alone). The arsenic-transformed cells showed a more rapid proliferation rate in complete medium than did control cells and also showed sustained proliferation in steroid-reduced medium. Although both control and CAsE-PE cells showed similar levels of androgen receptor (AR), androgens were less effective in stimulating cell proliferation and AR-related gene expression in CAsE-PE cells. For instance, dihydrotestosterone caused a 4.5-fold increase in prostate-specific antigen transcript in control cells but only a 1.5-fold increase in CAsE-PE cells. CAsE-PE cells also showed relatively low levels of growth stimulation by nonandrogen steroids, such as estradiol. Thus, arsenic-induced malignant transformation is associated with acquired androgen independence in human prostate cells. This acquired androgen independence was apparently not due to AR up-regulation, increased activity, or altered ligand specificity. The precise manner in which arsenic altered CAsE-PE growth and progression is undefined but may involve a bypass of AR involving direct stimulation of downstream signaling pathways.

Bile salts inhibit growth and induce apoptosis of culture human normal esophageal mucosal epithelial cells

To investigate the effect of six bile salts: glycocholate (GC), glycochenodeoxycholate (GCDC), glycodeoxycholate (GDC), taurocholate (TC), taurochenodeoxycholate (TCDC), taurodeoxycholate (TDC), and their mixture on cultured human normal esophageal mucosal epithelial cells. Human normal esophageal mucosal epithelial cells were cultured with serum-free keratinocyte medium.3-[4,5-Dimethylthiaolyl]-2,5-diphenyl-tetrazolium bromide assay was applied to the detection of cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay. Sub-G1 DNA fragmentations and early apoptotic cells were assayed by flow cytometry (FCM) with propidium iodide (PI) staining and annexin V-FITC conjugated with PI staining. Apoptotic DNA ladders on agarose gel electrophoresis were observed. Except for GC, GCDC, GDC, TC, TCDC, TDC and their mixture could initiate growth inhibition of esophageal mucosal epithelial cells in a dose- and time-dependent manner. TUNEL and FCM assays demonstrated that the bile salts at 500 mumol/L and their mixture at 1 500 micromol/L induced apoptosis except for GC. The percentage of sub-G1 detected by FCM with PI staining was 83.5% in cells treated with 500 micromol/L TC for 2 h, and 19.8%, 20.4%, 25.6%, 13.5%, and 75.8% in cells treated with 500 micromol/L GCDC, TCDC, GDC, TDC, and 1 500 micromol/L mixture for 24 h, respectively, which were higher than that of the control (1.5%). The percentage was 1.4% in cells with 500 micromol/L GC for 24 h. DNA ladders on agarose gel electrophoresis were seen in cells treated with 500 micromol/L TC for 2 h and 1 500 micromol/L mixture for 24 h. All GCDC, GDC, TC, TCDC, TDC and their mixture can inhibit growth and induce apoptosis of cultured human normal esophageal mucosal epithelial cells, but GC is well tolerated by the cells.

Differentiated Keratinocyte-Releasable Stratifin (14-3-3 Sigma) Stimulates MMP-1 Expression in Dermal Fibroblasts

Through the use of a keratinocyte/fibroblast co-culture system, we have recently identified a potent keratinocyte-derived anti-fibrogenic factor (KDAF) for dermal fibroblasts. A sequential chromatography of the active fractions of keratinocyte-conditioned medium (KCM) and peptide mapping of the candidate proteins identified KDAF as being the keratinocyte-releasable 14-3-3 sigma (14-3-3sigma) protein, which is also known as stratifin. In this study, we hypothesize that differentiated, but not proliferating, keratinocytes are the primary source of releasable 14-3-3sigma in conditioned medium. To address this hypothesis, in a longitudinal study, keratinocyte differentiation was induced by growing these cells in a medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% Dulbecco's modified eagle's medium without any additives for up to 20 d. When KCM was collected every other day and added to fibroblasts, the level of matrix metalloproteinase (MMP)-1 mRNA expression was markedly increased in fibroblasts receiving KCM and this increase was even greater in cells receiving conditioned media collected at later time points relative to that of controls. The results of a western blot analysis further showed a marked increase in the expression of 14-3-3sigma protein in keratinocytes grown in test medium from day 4 to day 10. This finding was consistent with the levels of 14-3-3sigma mRNA expression in differentiated keratinocytes. In contrast to a very high level of 14-3-3sigma mRNA expression seen in keratinocytes, fibroblasts that are highly responsive to14-3-3sigma were unable to express this factor. Interestingly, the level of 14-3-3sigma mRNA expression was markedly higher in keratinocytes co-cultured with fibroblasts relative to that of mono-cultured keratinocytes. In conclusion, this study provides evidence that keratinocytes express a high level of 14-3-3sigma at the levels of mRNA and protein. But the releasable form of 14-3-3sigma protein was only found in conditioned medium derived from differentiated keratinocytes. Further, our recently purified recombinant 14-3-3sigma protein mimics the collagenase stimulatory effect of KCM in dermal fibroblasts.

Isolation and Culture of Amelanotic Melanocytes from Human Hair Follicles

We report a method to establish amelanotic melanocytes (AMMC) in culture and we investigate the effects of various components in the culture medium. Normal human scalp from cadaver donors was transected 1 mm below the epidermis, and hair follicles in the remaining dermis were isolated by a two-step enzyme treatment. The individual hair follicles were washed exhaustively and suspensions of hair follicle cells were prepared and cultured in Eagle's minimum essential medium supplemented with 12-o-tetradecanoyl-phorbol-13-acetate (TPA), cholera toxin and keratinocyte serum-free medium (K-SFM). Geneticin was used to eliminate contaminating fibroblasts. Proliferation of AMMC was observed after addition of TPA and K-SFM including bovine pituitary extract (BPE) into the culture medium. Cell type was determined by staining with monoclonal antibodies, NKI/beteb and HMB-45, which recognize premelanosomal and melanosomal antigens, respectively. The AMMC were also examined using transmission electron microscopy. Treatment with geneticin eliminates the majority of fibroblasts and does not impair the growth of keratinocytes or AMMC. After contaminating fibroblasts and keratinocytes were removed, two distinct cell morphologies remained: (1) large, dendritic and deeply pigmented cells, which did not proliferate and which disappeared by the third passage, and (2) small bipolar cells, which initially were unpigmented and proliferated very rapidly. We observed that TPA at various concentrations stimulated the proliferation of the cells, and at high concentrations could induce the formation of multiple dendrites. K-SFM including BPE accelerated the proliferation of the cells in a dose-dependent manner. After passage 3, almost all cells expressed premelanosomal and melanosomal antigens, recognized by NKI/beteb and HMB-45, respectively. Active mitochondria, abundant rough endoplasmic reticulum, Golgi complexes, ribosomes and melannosomes (predominantly in stages I, II or III with some at stage IV in some AMMC) were observed ultrastructurally in the cytoplasm of the cultured cells.

Insulin suppresses collagenase stimulatory effect of stratifin in dermal fibroblasts

A delicate balance between synthesis and degradation of extracellular matrix (ECM) by matrix metalloproteinases (MMPs) is an essential feature of tissue remodeling. We have recently demonstrated that keratinocyte releasable stratifin, also known as 14-3-3 sigma protein, plays a critical role in modulating collagenase (MMP-1) mRNA expression in human dermal fibroblasts. In this study, we further characterized the collagenase stimulatory effect of stratifin in dermal fibroblasts and evaluated its effect in the presence and absence of insulin. Our data indicate that stratifin increases the expression of collagenase mRNA more than 20-fold in dermal fibroblasts, grown in either Dulbecco's modified Eagle's medium (DMEM) plus 2% or 10% fetal bovine serum (FBS). Collagenase stimulatory effect of stratifin was completely blocked, when fibroblasts were cultured in test medium consisting of 50% keratinocyte serum-free medium (KSFM) and 50% DMEM. The collagenase suppressive effect of test medium was directly proportional to the volume of KSFM used. As this medium contained insulin, we then evaluated the collagenase stimulatory effect of stratifin in dermal fibroblasts in the presence and absence of insulin. The results revealed that stratifin significantly increased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio, while insulin significantly decreased the expression of collagenase mRNA/18S (*p < 0.05, n = 3) ratio. The insulin inhibitory effect on collagenase mRNA expression was time and dose dependent. The maximal inhibitory effect of insulin was seen at 36 h post treatment. In conclusion, stratifin stimulates the expression of collagenase mRNA expression in dermal fibroblasts and this effect is suppressed by insulin treatment.

An in vitro outgrowth culture system for normal human keratinocytes

Background : Normal human epidermal keratinocytes usually proliferate in low-calcium and differentiate in high-calcium without a feeder layer, but they stop proliferating and differentiate at confluency even in low-calcium, serum-free medium. Objective : We speculated that this contact inhibition would be mediated in part by mechanical tension. To prove this, we created a new assay system. Methods : A 10 mm diameter cloning ring was put on the center of a 60 mm dish coated with type I collagen. Keratinocytes were plated in the ring and incubated for 4 h, then we had a circular epidermal monolayer sheet. We changed the mechanical tension by removing the ring and measured the diameter of the sheet under various conditions. Results : When we used keratinocyte-serum free medium (SFM) whose calcium concentration is below 0.1 mM as a medium, the keratinocytes in the perimeter migrated individually, and the keratinocytes in the center portion started differentiation. However, when we added calcium chloride to SFM (final concentration more than 0.5 mM), keratinocytes at the periphery showed marked lamellipodia without losing contact with the surrounding cells. These keratinocytes showed coordinate sheet-like outgrowth as a whole even in high concentrations of calcium. Conclusion : These results suggest that other than calcium concentration, change of the mechanical tension would be one of the factors that mediate proliferation or differentiation of keratinocytes and that this new assay can be useful in analyzing proliferation, differentiation, and migration of keratinocytes.

Proinflammatory cytokines upregulate expression of calprotectin (L1 protein, MRP-8/MRP-14) in cultured human keratinocytes.

Normal skin contains no epidermal calprotectin. In biopsies from various inflammatory skin disorders, however, this antimicrobial protein occurs in the cytoplasm of keratinocytes. To exclude the possibility of epidermal uptake of calprotectin from granulocytes and macrophages in diseased skin, we investigated whether cytokine-stimulated human keratinocytes can express calprotectin in vitro. Keratinocytes from healthy individuals were cultured in serum-free keratinocyte medium. The cells were stimulated with different cytokines [interferon (IFN)-gamma, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta, IL-10 and IL-13], both separately and in various combinations. Cytoplasmic protein levels of calprotectin were measured by an enzyme-linked immunosorbent assay performed on fixed adherent keratinocytes, and mRNA expression was determined by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Calprotectin was produced by cytokine-stimulated keratinocytes, especially in response to combinations of the proinflammatory cytokines, which showed an additive upregulatory effect. When expression of mRNA for the light (MRP-8) and heavy (MRP-14) calprotectin chain was determined by RT-PCR, their respective levels were shown to be increased four- to ninefold and three- to fivefold after 24 h of combined stimulation with IFN-gamma and TNF-alpha. The time course of calprotectin production showed no significant elevation for the first 16 h but then increased and peaked after 36 h. Cultured human keratinocytes stimulated with proinflammatory cytokines produce calprotectin, suggesting that epidermal expression of this antimicrobial protein in diseased skin reflects compartmentalized synthesis rather than uptake from dermal inflammatory cells.

Human dermal fibroblasts do not exhibit directional migration on collagen I in direct-current electric fields of physiological strength.

Endogenous electric fields are generated lateral to skin wounds, with the cathodal pole of the field residing in the center of the wound. These fields are thought to be an important mechanism in guiding the migration of keratinocytes and other cells into wounds to effect healing. In this work, human dermal fibroblasts were exposed to direct current electric fields of physiological strength, and their migrational behavior was quantitated. Only random migration of human dermal fibroblasts was observed in direct-current electric fields under conditions that support the directional migration of human epidermal keratinocytes. Additionally, neither the presence of serum nor serum plus additional Mg++ in the experimental medium supported directional migration. Migratory rates of fibroblasts varied depending on the experimental medium used: in serum-containing medium the average velocity was as low as 0.23 micro m/min, while in serum-free keratinocyte medium the average velocity was as high as 0.36 micro m/min. These studies suggest that dermal fibroblasts do not respond to the endogenous electric field of a wound, and use other migratory cues to direct their movement into the wound bed.

Sodium Pentosan Polysulfate Reduces Urothelial Responses to Inflammatory Stimuli Via an Indirect Mechanism

Sodium pentosan polysulfate has been promoted as a urothelial cytoprotective agent for treating interstitial cystitis. The nuclear transcription factor nuclear factor kappaB is thought to have a role in mediating the urothelial inflammatory response of interstitial cystitis. We further defined a possible cytoprotective effect of sodium pentosan polysulfate by characterizing the effect of the drug on the expression of nuclear factor kappaB. For cell culture human urothelial cells were incubated in various concentrations of sodium pentosan polysulfate for 16 hours in keratinocyte serum-free medium. They were subsequently treated with the known nuclear factor kappaB stimulants tumor necrosis factor-alpha, lipopolysaccaride (LPS) and double-stranded RNA (dsRNA). Each stimulant was then incubated with sodium pentosan polysulfate separately and the mixture was used to treat cultured urothelial cells. For electrophoretic mobility shift assay total cell extracts were prepared and run in electrophoretic mobility shift assays using a radiolabeled nuclear factor kappaB consensus sequence as a probe. Western blot analysis was done to assess nuclear factor kappaB activation by measuring degradation of the inhibitory subunit of the nuclear factor kappaB complex. Nuclear factor kappaB activation by tumor necrosis factor-alpha, LPS and dsRNA was unaltered when cultured cells were incubated in sodium pentosan polysulfate before treatment. In contrast, nuclear factor kappaB activation by LPS and dsRNA was suppressed when the stimulants were incubated with sodium pentosan polysulfate before cell treatment. This suppressive effect was confirmed by Western blot analysis. Sodium pentosan polysulfate may have a nonspecific effect against the viral (dsRNA) and bacterial (LPS) activation of nuclear factor kappaB. The observed clinical effect of sodium pentosan polysulfate may be mediated by nonspecific binding of sodium pentosan polysulfate molecules and the inflammatory stimulants of urothelial activation. These findings suggest a mechanism of action for sodium pentosan polysulfate that occurs in the urine rather than at the mucosal membrane by direct interaction of the drug with potential interstitial cystitis inducing inflammatory agents.

Isolation, cultivation, and differentiation of normal human epidermal keratinocytes in serum-free medium.

The outer layer of human skin is the epidermis. More than 90% of the epidermal cells are keratinocytes, which originate from stem cells, located in the basal cell layer (Stratum basale). Keratinocytes in the basal layer divide, differentiate, and migrate to the outer layer of the epidermis, the horny layer (Stratum corneum). The horny layer consists of terminally differentiated keratinocytes (corneocytes) embedded in multilamellar lipid sheets (). Finally, corneocytes are shed from the surface of the skin by desquamation. Keratinocytes are an ideal target for gene therapy (,) to treat desquamation disorders (,). In addition, keratinocytes are secretory cells (), which opens the possibility for systemic delivery (,).

Toxicity of natural tear substitutes in a fully defined culture model of human corneal epithelial cells

PURPOSE. Serum and saliva have recently been advocated as natural tear substitutes for intractable aqueous-deficient dry eyes, but the effects of these fluids on corneal epithelium have not been well characterized. A laboratory study was performed in a defined test model to compare the toxicity of natural and pharmaceutical tear substitutes and to identify potentially toxic factors in natural tear substitutes, such as amylase, hypotonicity, and variations in preparation. METHODS. Primary human corneal epithelial cells were cultured with defined keratinocyte serum-free medium. The cells were incubated with hypromellose (hydroxypropylmethylcellulose 0.3%) with and without benzalkonium chloride 0.01%, saliva with differing osmolalities, 100% serum, and 50% serum (1:1 vol/vol with chloramphenicol 0.5%) for varying times and concentrations. Toxicity was examined in four ways. Microvillous density was assessed with scanning electron microscopy. Cell membrane permeability and intracellular esterase activity were analyzed after staining with fluorescent calcein-AM/ ethidium homodimer and cellular adenosine triphosphate (ATP) was quantified using a luciferin-luciferase‐ based assay. RESULTS. The toxicity ranking of the tear substitutes correlated in all assays. The ATP assay was the most sensitive, followed by ethidium cell permeability, and finally the esterase activity. Preserved hypromellose was more toxic than the unpreserved preparation. Among natural tear substitutes, natural saliva was most toxic. Isotonic saliva and 50% serum were of similar toxicity, and 100% serum was least toxic. Natural tear substitutes were— except for natural saliva—less toxic than unpreserved hypromellose. Hypotonicity, but not amylase, was the major toxic effect associated with saliva. The dilution of serum with chloramphenicol induced toxicity. CONCLUSIONS. This is the first toxicity study using human primary corneal epithelial cells cultured under fully defined conditions as an in vitro model. Cellular ATP is a sensitive parameter for quantifying toxicity. Isotonic saliva and serum offer greater therapeutic potential for severely aqueous-deficient dry eyes than do pharmaceutical tear substitutes. (Invest Ophthalmol Vis Sci. 2001;42:948 ‐956)

Expansion and long-term culture of differentiated normal rat urothelial cells in vitro.

The objective of this study is to establish a reliable cell culture system for the long-term culture of rat urothelial cells (RUC), in which the cells multiply in vitro and form stratified polarized urothelium. Urothelial cells were harvested by the enzymatic digestion of the urothelium exposed by the eversion of resected rat bladders. Primary cultures were initiated in keratinocyte serum-free medium (KSFM) for selective proliferation of urothelial cells. Subsequently, the cells were propagated in a mixture of conditioned medium (CM) derived from Swiss 3T3 cell culture supernatant and KSFM (CM-KSFM). Mean population doubling time was 13.8 +/- 0.9 h. RUC were successfully maintained for 18 passages over a period of 4-5 mo. Detailed investigations of culture conditions showed that CM-KSFM yielded a differentiated multilayer structure. The stratified urothelial sheets measuring 4 x 6 cm2 could be formed and then detached using dispase. Cytokeratin pattern in both the cultured urothelial monolayer and engineered stratified layers was similar to those seen in vivo, as assessed with monoclonal antibody against cytokeratin 17. Ultrastructural morphology showed microvilli, basal cell layer, and desmosomes between adjacent cells in the stratified urothelium.

The influence of crosslinking agents and diamines on the pore size, morphology and the biological stability of collagen sponges and their effect on cell penetration through the sponge matrix.

Artificial skin substitutes based on autologous keratinocytes cultured on collagen substrata are being developed for treating patients with severe burns. The properties of the collagen substrate can be manipulated, for example, by crosslinking, to optimize desirable properties such as cell growth and penetration into the substrate, biological stability and mechanical strength. Collagen sponges crosslinked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDAC) and the diamine, diaminohexane, were used to determine the effect of crosslinking on pore size and morphology, on the stability of the crosslinked sponges both in cell culture media and during incubation with collagenase, and on the penetration of keratinocytes and fibroblasts through the sponge matrix. Crosslinking of the sponges reduced the pore size, particularly at the surface, and altered sponge morphology. After crosslinking the collagen fibers were thinner, and appeared lacy and delicate. Crosslinking also influenced sponge stability. In keratinocyte serum-free medium the pore size of plain collagen sponges increased with increasing incubation time, and crosslinking appeared to prevent this, and may have stabilized sponge structure. Incubation in serum-containing Dulbecco's minimum essential medium caused a marked reduction in pore size in both plain collagen and crosslinked collagen sponges. Crosslinking did not appear to influence this cell-free contraction of collagen sponges. Treatment of sponges with EDAC markedly increased the resistance of sponges to collagenase digestion. The penetration of both keratinocytes and fibroblasts was retarded by crosslinking the sponges. Fibroblasts penetrated through the sponges to a greater extent than keratinocytes, and their proliferation rate was faster. The total number of cells populating the crosslinked sponges after 10 days culture was approximately 50% of that on untreated collagen sponges. The mechanism responsible for this effect was different with the two crosslinkers used. Diaminohexane appeared to inhibit cell growth, whereas EDAC may have caused a decrease in cell adhesion to the sponges, without an apparent inhibition of growth rate. In terms of morphology, fibroblasts were elongated to a greater extent on crosslinked sponges, and alligned themselves along the collagen fibers. Keratinocytes grew in colonies on untreated sponges, but on crosslinked sponges they grew in isolation, with minimal cell-cell interactions. It may be necessary to reach a compromise to obtain the best combination of properties for using collagen sponges as substrata for artificial skin substitutes.

Multi-layered periodontal pocket epithelium reconstituted in vitro: histology and cytokeratin profiles.

In order to study inter-individual differences in bacterial adhesion/invasion of periodontal tissues, an in vitro model for culturing multi-layered pocket epithelium without feeder layers or stromal equivalents (including the evaluation of their cytokeratin profiles) was developed. Pocket epithelium was collected and grown until confluent in Falcon flasks using keratinocyte-serum free medium (KSFM), without a feeder layer. In the second passage, oral keratinocytes were re-grown in a 2 compartment system using either a clear polyester (transwell-clear [TCL]) or a collagen (transwell-col [TCO]) membrane as culture surface. After the first week, the calcium concentration was raised to 1.2 mM and in half the wells, the KSFM was supplemented with 10% fetal calf serum (FCS). Histology and immunohistochemistry were performed after 1, 2, and 3 weeks of additional growth. In general, all conditions resulted in a structured epithelium consisting of 3 to 5 layers, but important differences were observed between the membrane types and between the media. CK4 was rarely and only lightly expressed while CK18 and 19 (characteristic of junctional epithelium) were very strongly expressed in the older (2 and 3 weeks) cultures. CK13 and 14 (characteristic of any stratifiable epithelial cell) also tended to increase over time; CK13 seemed to be stronger in KSFM with FCS while the contrary was true for CK14. The multi-layer created by the combination TCL/KSFM + 10% FCS resembled a junctional epithelium most, while that grown on TCO without FCS mimicked the sulcular epithelium. It seems possible to create a histiotypic culture resembling either periodontal pocket or junctional epithelium without the use of stromal equivalents or feeder layers which make this approach more cumbersome. This multi-layered culture offers a model to investigate the permeability of pocket epithelium and the adhesion and penetration of bacteria under well-defined environmental conditions.

Generation of papillomavirus-immortalized cell lines from normal human ectocervical, endocervical, and vaginal epithelium that maintain expression of tissue-specific differentiation proteins.

We have established and characterized three cell lines from normal human vaginal, ectocervical, and endocervical epithelia immortalized by expression of human papillomavirus 16/E6E7. The lines (VK2/E6E7, Ect1/E6E7, and End1/E6E7) displayed distinctive morphologies at the level of light microscopy when cultured in calcium-supplemented (0.4 mM) keratinocyte serum-free medium and maintained a stable phenotype after more than 1 yr of continuous passage. They were compared to primary cell cultures and epithelial cells in sections of the respective native tissues for expression of epithelial differentiation proteins. All cell lines expressed cytokeratin (CK) 8, CK18, and CK19, and some cells in all three cell lines expressed CK16, involucrin, and the secretory component of the polymeric immunoglobulin receptor. The vaginal and ectocervical cell lines expressed CK10 and CK13, whereas the endocervical line did not. With the exception of CK8 and CK18 expression, the morphological and immunocytochemical characteristics of the immortalized lines closely resembled those of their respective tissues of origin and primary cultures, and all differed significantly from the HeLa cervical adenocarcinoma cell line. These new cell lines may provide the basis for valid, reproducible in vitro models for studies on cervicovaginal physiology and infections and for testing pharmacological agents for intravaginal application.

A technique for the culture of Barrett's oesophageal cells.

Establishment of cells in tissue culture from Barrett's columnar epithelium has been difficult. The aim of this study was to develop a successful tissue culture method employing a serum-free medium for cultivation of Barrett's epithelial cells. Fragments of Barrett's mucosal tissue were explanted in a 3:1 mixture of Dulbecco's modification of Eagle's medium and Ham's F12, to initiate the outgrowth of epithelial cells. Subsequently, a commercial serum-free medium (formulated for the growth of keratinocytes) was used for the propagation of Barrett's oesophagus cells without fibroblast growth. Cells established in culture retained their epithelial morphology, stained positive for cytokeratin, and contained Alcian blue (pH 2.5) and periodic acid-Schiff reagent-positive/diastase-resistant vacuoles, confirming their origin from Barrett's epithelium. Electron microscopy showed tonofilaments, microvilli and desmosomes. Coating the surface of culture vessels was not required and four cell strains could be passaged up to 20 times with no fibroblast growth, in the keratinocyte serum-free medium.

Cultivation, Serial Transfer, and Differentiation of Epidermal Keratinocytes in Serum-Free Medium

We describe serum-free culture conditions for human epidermal keratinocytes using lethally treated 3T3 cells as feeder layers and normal Ca++concentrations (1.2 mM), in a DMEM/F12-Ham nutrient mixture supplemented with several additives, and 10 mg/ml bovine serum albumin instead of animal serum. Keratinocytes were serially grown to 15–18 cell generations (4 subcultivations) and formed a stratified squamous epithelium that could be detached as a graftable epithelial sheet. EGF and TGFα significantly increased keratinocyte proliferation under these conditions; EGF reduced the expression of keratin K1, which is specific for stratified and terminally differentiated epidermal keratinocytes. In contrast with previous reports, the serum-free medium we describe here supports serial growth and normal differentiation of human epidermal keratinocytes, and the formation of graftable stratified epithelia; it also supports the assay of a variety of cytokines or compounds that modulate epidermal keratinocyte proliferation and differentiation.