3D profilometry and cell viability studies for drug response screening.

In vitro tests for assessing cell viability and drug response are widely employed for determining cytotoxicity of drugs, chemicals, or material substrates. These assays have some advantages, such as speed, reduced cost, and potential for automation. However, since these tests are often run with a huge amount of cells, the characteristic properties of a single cell can be masked leading to a lack of the diagnostic features of these assays. Vital processes as proliferation and cell death (either necrosis or apoptosis) are associated to drastic changes of volume and surface analysis techniques like 3D optical scanning profilometry allow noninvasive and nondestructive approach with fast detection and good resolution at nano-microscale. Here, we demonstrate how coupling noninvasive morphological surface analysis techniques with well assessed biochemical methods can help to establish the relationship between the modifications on cellular viability induced by precursors of proliferation and cell death and variations on cell volume induced by these treatments. The proposed approach has demonstrated improved efficiency on the assessment of inductive changes on tumoral cells in comparison to non-tumoral cells upon administration of proliferative nontoxic or cytotoxic substances like chemotherapeutics.

Human eccrine sweat gland cells can reconstitute a stratified epidermis.

Eccrine sweat glands are generally considered to be a possible epidermal stem cell source. Here we compared the multilayered epithelia formed by epidermal keratinocytes and those formed by eccrine sweat gland cells. We demonstrated both in vitro and in vivo the capability of human eccrine sweat gland cells to form a stratified interfollicular epidermis substitute on collagen hydrogels. This is substantiated by the following findings: (1) a stratified epidermis consisting of 10-12 cell layers is formed by sweat gland cells; (2) a distinct stratum corneum develops and is maintained after transplantation onto immuno-incompetent rats; (3) proteins such as filaggrin, loricrin, involucrin, envoplakin, periplakin, and transglutaminases I and III match with the pattern of the normal human skin; (4) junctional complexes and hemidesmosomes are readily and regularly established; (5) cell proliferation in the basal layer reaches homeostatic levels; (6) the sweat gland-derived epidermis is anchored by hemidesmosomes within a well-developed basal lamina; and (7) palmo-plantar or mucosal markers are not expressed in the sweat gland-derived epidermis. These data suggest that human eccrine sweat glands are an additional source of keratinocytes that can generate a stratified epidermis. Our findings raise the question of the extent to which the human skin is repaired and/or permanently renewed by eccrine sweat gland cells.

Characterization of mouse 3T3-swiss albino cells available in Japan: necessity of quality control when used as feeders.

Mouse 3T3-Swiss albino cells are widely used as feeder cells to culture the human epidermis for the treatment of burns. To minimize the risk of xenoinfection, quality control of the feeder cells is required in the Japanese guidelines on regenerative medicine using feeder cells. We characterized three lots of 3T3-Swiss albino cells that are publicly or commercially available in Japan. One lot, which propagated more rapidly than the other two without showing typical contact inhibition, was found to release endogenous murine leukemia virus upon iododeoxyuridine-treatment. Southern blotting of restriction fragments showed that the rapidly growing lot consisted of genetically altered cells that had probably emerged during the passages. The data support the guidelines that recommend the quality control of each lot of 3T3-Swiss albino cells if they are to be used clinically.

Pivotal role of the mineralocorticoid receptor in corticosteroid-induced adipogenesis.

In addition to their role in controlling water and salt homeostasis, recent work suggests that aldosterone and mineralocorticoid receptors (MR) may be involved in adipocyte biology. This is of particular relevance given the role of MR as a high-affinity receptor for both mineralocorticoids and glucocorticoids. We have thus examined the effect of aldosterone and MR on white adipose cell differentiation. When cells are cultured in a steroid-free medium, aldosterone promotes acquisition of the adipose phenotype of 3T3-L1 and 3T3-F442A cells in a time-, dose-, and MR-dependent manner. In contrast, late and long-term exposure to dexamethasone inhibits adipocyte terminal maturation. The aldosterone effect on adipose maturation was accompanied by induction of PPARgamma mRNA expression, which was blocked by the MR antagonist spironolactone. Under permissive culture conditions, specific MR down-regulation by siRNAs markedly inhibited 3T3-L1 differentiation by interfering with the transcriptional control of adipogenesis, an effect not mimicked by specific inactivation of the glucocorticoid receptor. These results demonstrate that MR represents an important proadipogenic transcription factor that may mediate both aldosterone and glucocorticoid effects on adipose tissue development. MR thus may be of pathophysiological relevance to the development of obesity and the metabolic syndrome.