Comparison of Human Dermal Fibroblasts and HaCat Cells Cultured in Medium with or without Serum via a Generic Tissue Engineering Research Platform

Abstract

A generic research platform with 2-dimensional (2D) cell culture technology, a 3-dimensional (3D) in vitro tissue model, and a scaled-down cell culture and imaging system in between, was utilized to address the problematic issues associated with the use of serum in skin tissue engineering. Human dermal fibroblasts (HDFs) and immortalized keratinocytes (HaCat cells) mono- or co-cultured in serum or serum-free medium were compared and analyzed via the platform. It was demonstrated that serum depletion had significant influence on the attachment of HaCat cells onto tissue culture plastic (TCP), porous substrates and cellulosic scaffolds, which was further enhanced by the pre-seeded HDFs. The complex structures formed by the HDFs colonized within the porous substrates and scaffolds not only prevented the seeded HaCat cells from filtering through the open pores, but also acted as cellular substrates for HaCat cells to attach onto. When mono-cultured on TCP, both HDFs and HaCat cells were less proliferative in medium without serum than with serum. However, both cell types were successfully co-cultured in 2D using serum-free medium if the initial cell seeding density was higher than 80,000 cells/cm2 (with 1:1 ratio). Based on the results from 2D cultures, co-culture of both cell types on modular substrates with small open pores (125 μm) and cellulosic scaffolds with open pores of varying sizes (50–300 µm) were then conducted successfully in serum-free medium. This study demonstrated that the generic research platform had great potential for in-depth understanding of HDFs and HaCat cells cultivated in serum-free medium, which could inform the processes for manufacturing skin cells or tissues for clinical applications.

Adding a new dimension: Multi-level structure and organization of mixed-species Pseudomonas aeruginosa and Staphylococcus aureus biofilms in a 4-D wound microenvironment

“…The co-cultured scaffolds showed an average of 140േ16 cells (both HaCaT and HDFa) in a visualized area of 581.2 X 581.2 ߤm, consisting of 88േ16 HaCaT cells and 52േ4 HDFa cells. This results in 13000േ1500 cells in the well (culturable surface area of 0.32 X 10 8 ߤm 2 ; n=2, two biological replicates with two fields of view per well, error represents SEM), which based on the average cell area for HaCaT and HDFa cells, 3000 ߤm 2 [54-57] and 4000 ߤm 2 [54][55][56][57] respectively, corresponds to a coverage of close to 100% of the well surface [58,59]. It is important to note that in the composite 4-D wound microenvironment, the IVWM would provide matrix elements (collagen, fibrinogen, fibronectin), which would be present between, and in close association, with the host cells.…”

To Serum or Not to Serum: Reduced-serum Based Approaches for Contact-based Co-culture of Fibroblasts and Keratinocytes for Wound Bed Studies

“…When FGF is added to MEM at a concentration of 1X, the resulting media contains 2% FBS; KGF is serum-free. These approaches result in effective concentrations of 1-2% FBS, representing a 5-10-fold reduction as compared with previous protocols[7,8,10] (using up to 10% FBS).…”

“…Interactions between these two cell types critically impact wound bed structure and functions, including cellular architecture, cell migration and wound closure [5,6]. Contact-based co-culture of human fibroblasts and keratinocytes has been challenging, with previous protocols employing high concentrations of animal serum (up to 10%), cell feeder systems and a range of cell-specific growth factors [7][8][9][10]. These approaches are not only tedious and laborintensive, but the usage of high-serum concentrations poses a range of well-known technical and scientific limitations [11][12][13], including variable effects based on different cell types.…”

“…These approaches are not only tedious and laborintensive, but the usage of high-serum concentrations poses a range of well-known technical and scientific limitations [11][12][13], including variable effects based on different cell types. For example, in the context of keratinocytes and fibroblasts, the presence of serum is observed to influence the attachment and proliferation [7] of the two cell types when cultured together. In one study [7], keratinocytes (HaCaT) co-cultured with fibroblasts (HDFa) in the presence of 10% fetal bovine serum, were seen to attach poorly to tissue culture plastic substrates; attachment was significantly higher under serum-free conditions.…”

“…For example, in the context of keratinocytes and fibroblasts, the presence of serum is observed to influence the attachment and proliferation [7] of the two cell types when cultured together. In one study [7], keratinocytes (HaCaT) co-cultured with fibroblasts (HDFa) in the presence of 10% fetal bovine serum, were seen to attach poorly to tissue culture plastic substrates; attachment was significantly higher under serum-free conditions. On the other hand, alternatives such as serum-free media or synthetic formulations either do not support the robust co-culture of fibroblasts and keratinocytes; fibroblasts have been shown to display impaired proliferative capacity under serum-free conditions [7], or require substantial optimization and are cost-intensive [11,13].…”