Effect of Multiple-Cycle Collections of Conditioned Media from Different Cell Sources towards Fibroblasts in In Vitro Wound Healing Model

Abstract

Conditioned media refers to a collection of the used cell culture media. The goal of this study was to evaluate the possible impacts of different conditioned media collected across a number of cycles on the fibroblast proliferation, migration, and profiles of protein release. Human dermal fibroblast (HDF) cells and Wharton jelly mesenchymal stem cells (WJMSC) were cultured and incubated for 3 days prior to being harvested as cycle-1 using the serum-free media F12:DMEM and DMEM, respectively. The procedures were repeatedly carried out until the fifth cycle of conditioned media collection. An in-vitro scratch assay was conducted to measure the effectiveness of wound healing. Collagen hydrogel was combined separately with both the Wharton jelly-conditioned medium (WJCM) and the dermal fibroblast-conditioned medium (DFCM) in order to evaluate the protein release profile. The conditioned medium from many cycles had a lower level of fibroblast attachment than the control (complete medium); however, the growth rate increased from 100 to 250 h^-1, when supplemented with a conditioned medium collected from multiple cycles. The wound scratch assay showed that fibroblast cell migration was significantly increased by repeating cycles up to cycle-5 of DFCM, reaching 98.73 ± 1.11%. This was faster than the rate of migration observed in the cycle-5 of the WJCM group, which was 27.45 ± 5.55%. Collagen hydrogel from multiple cycles of DFCM and WJCM had a similar protein release profile. These findings demonstrate the potential for employing repeated cycles of DFCM- and WJCM-released proteins with collagen hydrogel for applications in wound healing.

Keywords: Wharton jelly; conditioned medium; dermal fibroblast; secretory protein; wound healing.

Figure 1

(A) Cell morphology during collection of multiple-cycle conditioned media, and (B) protein concentrations of collection multiple cycles of different cell sources (DFCM and WJCM). Data are expressed in mean ± standard deviation (SD), N = 3; n = 3. * p < 0.05 compared to the cycle-1 group.

Figure 2

The visualisation and separate proteins of concentrated multiple cycles of (A) DFCM and (B) WJCM in comparison to the respective controls by 1D SDS-PAGE.

Figure 3

The morphology of fibroblast cells after supplementation with (A) DFCM and (B) WJCM throughout 72 h treatment.

Figure 4

(A) The effect of multiple cycles of both DFCM and WJCM on fibroblast attachment. The data representation of three different experiments and expressed as mean ± SD; * p < 0.05 when compared with control (completed medium). (B) The effect of multiple cycles of both DFCM and WJCM on fibroblast growth rate. The data representation of three different experiments and expressed as mean ± SD; * p < 0.05 when compared with control (completed medium).

Figure 5

The effect of DFCM and WJCM on fibroblast proliferation. (A) Representative images of immunocytochemistry staining of fibroblasts supplemented by multiple cycles DFCM and WJCM, with anti-Ki67 (red) and nuclear staining (blue). The yellow arrow indicates positive expression of proliferative cells with anti-Ki67. Scale bar is 100 µm. Magnification 20×. (B) Quantitative evaluation (in percentage) of proliferative cells for both treated groups (DFCM and WJCM).

Figure 6

The effect of multiple cycles of (A) DFCM and (B) WJCM on the fibroblast migration for 48 h. The representative images of fibroblast migration treated with 1×–5× cycles of DFCM and WJCM. Yellow line indicates cell movement. Scale bar is 100 µm. Healing progression of multiple cycles (C) DFCM and (D) WJCM. Data were shown as mean ± SD obtained from triplicate experiments. The asterisks represent * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 7

(A) Gross morphology and general structure of collagen hydrogel fortified with DFCM- and WJCM and (B) cumulative amount of protein released from collagen hydrogel over 24 h period.