title: Effects of microgravity on growing cultured skin constructs.
abstract: Understanding the cellular, chemical, and physical responses of cells to stimuli is critical to successfully engineering tissue. The effect of culturing a living skin equivalent (LSE) in a submerged microgravity environment was investigated. LSEs were developed by culturing normal human epidermal keratinocyte (NHEK) on a submerged fibroblast and type 1 collagen gel matrix. Once formed, LSEs were brought up to the air/liquid interface, and after 4 days, the cultures were maintained in either (a) a normal air/liquid interface (S), (b) resubmerged in media (R), (c) folded on themselves to enclose the keratinized layer (F/R), or (d) cut into 2-4-mm fragments and suspended in a state of microgravity in a NASA-designed bioreactor (B). All groups were cultured for an average of 3 additional days. LSEs were processed for histologic evaluation. Skin cells were stained for cytokeratin to evaluate function. Images were digitally captured and processed for analysis. Parameters, including epithelial thickness, cellular areas, nuclear number, nuclear area, cytoplasmic area, and stained cytokeratin areas were measured. Removing the air/media interface significantly increased the number of NHEKs present in the skin; microgravity greatly enhanced this effect (p < 0.0001). No significant difference in cellular function as measured by protein expression [stained cytokeratin area (micro(2)) per cell] was found among the groups, though the ratio of nuclear area was significantly increased in all three groups as compared to the S group (p = 0.00227). In the case of the R and F/R groups, this appears due to the loss of the NHEK layer associated with those groups. Additionally, significant nuclear hypertrophy was demonstrated in the B group (p < 0.0001), and cellular hyperplasia was measured in all submerged groups as compared to static (p < 0.0001). Elimination of the air/liquid interface enhanced proliferation of keratinocytes. This effect was further enhanced in the presence of microgravity. No significant effect on cell function was noted with the use of this microgravity environment. We hypothesize that the increased epidermal contact plays a role in this proliferation. Microgravity is also associated with nuclear and cellular hypertrophy over and above that of the submersion methods.
authors.affiliation: Cooper Health System, UMDNJ/Robert Wood Johnson Medical Center, Camden, NJ 08103, USA. doolinej@umdnj.edu
authors.forename: E J
authors.initials: EJ
authors.lastname: Doolin
authors.forename: B
authors.initials: B
authors.lastname: Geldziler
authors.forename: L
authors.initials: L
authors.lastname: Strande
authors.forename: M
authors.initials: M
authors.lastname: Kain
authors.forename: C
authors.initials: C
authors.lastname: Hewitt
mesh_terms: D000388:Air; D015415:Biomarkers / Q000032:analysis; D019149:Bioreactors; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division; D002467:Cell Nucleus / Q000648:ultrastructure; D018920:Coculture Techniques; D003094:Collagen; D003470:Culture Media; D000078404:Epidermal Cells; D005347:Fibroblasts / Q000166:cytology; D005782:Gels; D006801:Humans; D007101:Immersion; D015603:Keratinocytes / Q000166:cytology*; D007633:Keratins / Q000032:analysis; D008297:Male; D014893:Weightlessness
chemical_list: D015415:Biomarkers; D003470:Culture Media; D005782:Gels; D007633:Keratins; D003094:Collagen