CES have been shown to be mainly composed of basal and spinous keratinocytes adhered via desmosomes and other adhesion proteins such as E-cadherin and-catenin [13]. of keratins, involucrin, and p63. There were no differences in the epidermal layer or protein expression between L-CES Rabbit Polyclonal to CYSLTR1 and F-CES, and both CES were comparable to fresh CES. TGF-, EGF, VEGF, IL-1, and MMPs were detected in L-CES at levels similar to those in F-CES. In a mouse study, wounds treated with L-CES or F-CES completely healed at least 4 days faster than untreated wounds. CES-treated wounds completely healed by day 10, while the untreated wounds did not heal by day 14. Masson’s trichrome staining showed that collagen deposition in the CES-treated wounds was highly increased in the dermis of the wound center compared to that in the control wounds. Thus, this study demonstrates that L-CES is as clinically effective as F-CES for wound treatment. == 1. Introduction == CES have been used to treat cutaneous wounds such as burns and ulcers since the technologies to culture keratinocytes and to detach CES from the culture vessels were established [13]. It was shown in the late 1980s that CES of allogeneic keratinocytes, unlike their autologous counterpart, are not permanent, instead serving as a temporary biological dressing that releases proteins involved in the proliferation and migration of keratinocytes and fibroblasts. Proteins that act in concert to promote wound healing include growth factors such as bFGF, TGF-, TGF-, EGF, VEGF, and PDGF, cytokines such as IL-1, IL-6, and IL-8, and extracellular matrix proteins such as collagen, laminin, tenascin, and fibronectin [46]. Allogeneic CES, prepared from a cell bank established from an infant skin biopsy, have advantages over autologous CES in that the time taken to obtain CES from primary culture is much STL127705 shorter compared to autologous CES. In order for the allogeneic CES to be readily available, however, it is necessary to store them for some period of time without compromising their biological nature. Studies have STL127705 demonstrated that the F-CES are as effective as freshly prepared CES in treating partial-thickness wounds [7,8]. Furthermore, L-CES were also shown to successfully treat chronic leg ulcers [9,10]. Because lyophilization seems to provide a convenient and cost-effective way to prepare ready-made CES on a large scale, we were curious to know whether the wound-healing effects of L-CES are comparable to F-CES. == 2. Materials and Methods == == 2.1. Preparation of F-CES and L-CES == Human keratinocytes were cultured as described in Barrandon and Green to form a sheet [11] that was detached from the culture flask using dispase [2]. For cryopreservation, the resulting CES were treated with a cryoprotectant containing human serum albumin in DMEM and then frozen at 70C. Before lyophilization, CES were treated with STL127705 a lyoprotectant of 5% polyethylene glycol (PEG), 1% trehalose, and cryoprotectant and then frozen at 40C. More than 12 hours later, lyophilization was performed through a gradual increase in temperature at the rate of 1C per minute from 40C to 25C under vacuum pressure of less than 10 mTorr in a lyophilizer (Ilshin Biobase, Seoul, South Korea). == 2.2. Microstructural Observation of CES == The sheet surface of CES was observed using a field-emission scanning electron microscope (FE-SEM) (SUPRA 55VP, Carl Zeiss, Oberkochen, Germany). The cross sections of CES were examined for cell-cell junctions with a transmission electron microscope (TEM) (JEM1010, JEOL, Akishima, Tokyo, Japan). == 2.3. Immunofluorescence Staining (IF) == Monoclonal antibodies raised against K1 (Abcam, Cambridge, MA, USA), K14 (AbD Serotec, Raleigh, NC, USA), involucrin (Sigma-Aldrich, St. Louis, MO, USA), and p63 (BD Pharmingen, San Jose, CA, USA) were used with secondary antibodies including anti-mouse IgG conjugated with either FITC (Jackson Immuno Research Laboratories Inc., West Grove, PA, USA) or rhodamine (Invitrogen, Grand Island, NY, USA). A mounting medium containing DAPI (Vector, Burlingame, CA, USA) was used for nuclear staining. IF images were observed with an Anaxioskop 40 FL Microscope (Carl Zeiss). == 2.4. ELISA Assay == Protein extracts from CES were prepared by repeated freezing and thawing followed by homogenization. Commercially available ELISA kits were used according to the.