HEC Cell Viability and Collagen Production Study

1. OBJECTIVE

The purpose of this study was to evaluate the ability of the Hydrolytic Enzyme Complex to improve cell viability and to stimulate the production of Type I collagen.

2. PROCEDURE

2.1 Preparation of Fibroblasts

Fibroblasts were seeded into the individual wells of a 24-well plate in 0.5 ml of Fibroblast Growth Media (FGM) and incubated overnight at 37+2oC and 5+1% CO2. On the following day the media was removed via aspiration to eliminate any non-adherent cells and replaced with 0.5 ml of fresh FGM. The cells were grown until confluent, with a media change every 48 to 72 hours. Upon reaching confluency the cells were treated for 24 hours with DMEM supplemented with 1.5% FBS to wash out any effects from the growth factors included in the normal culture media. After this 24-hour wash out period the cells were treated with the test materials at the specified concentrations dissolved in FGM with 1.5% FBS. TGF-B (50 ng/ml) was used as a positive control for collagen and elastin, while 100 uM DbcAMP was used as a positive control for hyaluronic acid. Untreated cells (negative controls) just received DMEM with 1.5% FBS. The cells were incubated for 48 hours and at the end of the incubation period cell culture medium was collected and either stored frozen (-75°C) or assayed immediately. Materials were tested in triplicate.

2.2 MTT Assay

After the 2-day incubation, the cell culture medium was removed (see above) and the fibroblasts were washed twice with PBS to remove any remaining test material. After the final wash, 500 μl of DMEM supplemented with 0.5 mg/ml MTT was added to each well and the cells were incubated for 1 hour at 37+2oC and 5+1% CO2. After the incubation, the DMEM/MTT solution was removed, and the cells were washed again once with PBS and then 0.5 ml of isopropyl alcohol was added to the well to extract the purple formazin crystals. Two hundred microliters of the isopropyl extracts were transferred to a 96-well plate and the plate was read at 540 nm using isopropyl alcohol as a blank. The mean MTT absorbance value for the negative control cells was calculated and used to represent 100% cell viability. The individual MTT values from the cells undergoing the various treatments were then divided by the mean value for the negative control cells and expressed as a percent to determine the change in cell viability caused by each treatment.

2.3 Collagen Production Assay

A series of type I C-peptide standards was prepared ranging from 0 ng/ml to 640 ng/ml. Next, an ELISA microplate was prepared by removing any unneeded strips from the plate frame followed by the addition of 100 μl of peroxidase-labeled anti procollagen type I-C peptide antibody to each well used in the assay. Twenty (20) μl of either sample (collected tissue culture media) or standard was then added to appropriate wells, and the microplate was covered and allowed to incubate for 3 ± _0.25 hours at 37°C. After the incubation the wells were aspirated and washed three times with 400 μl of wash buffer. After the last wash was removed 100 μl of peroxidase substrate solution (hydrogen peroxide + tetramethylbenzidine as a chromagen) was added to each well and the plate was incubated for 15 ± _5 minutes at room temperature. After the incubation 100 μl of stop solution (1 N sulfuric acid) was added to each well and the plate was read using a microplate reader at 450 nm.

3.0 Results

The Hydrolytic Enzyme Complex was found to significantly improve cell viability (Graph 1) and increase the production of Type I collagen (Graph 2) when compared to untreated cells.

Graph 1. Cell Viability Study (MTT Assay)

Graph 2. Type I Collagen Production Assay

Disclaimer: This document is a summary of a Hydrolytic Enzyme Complex clinical trial performed by an independent third-party clinical testing facility. We believe the information provided here is correct but past performance is not necessarily indicative of future results.