In the proliferative phase of wound healing, the wound bed is progressively filled with granulation tissue (1), followed by re-epithelialization to restore surface integrity (2). Granulation tissue consists of, among other components, fibroblasts from the dermis, which proliferate and migrate into the wound bed (3). Fibroblasts secrete signaling factors that recruit adjacent epidermal keratinocytes from the wound edge and epithelial appendages (3,4) (Fig. 1A). Migration of keratinocytes and fibroblasts results in regions of direct contact between the two cell types (5,6). Interactions between these two cell types critically impacts wound bed structure and functions, including cellular architecture, cell migration and wound closure (6–9). Therefore, it is important to develop approaches that not only enable the co-culture of these two cell types in direct contact with each other but can also be leveraged to study important features and functions of the wound bed.
Contact-based co-culture of human dermal fibroblasts (fibroblasts) and epidermal keratinocytes (keratinocytes) has been a long-standing and challenging issue in the field of wound and skin studies (10–19) (Table 1). Fibroblasts and keratinocytes (primary cells and cell lines), as well as cell lines, require a host of growth factors and supplements to support their in vitro proliferation (20–23), the concentrations and compositions of which vary across the two cell types. Animal serum (fetal bovine serum (FBS) or fetal calf serum (FCS)) contains a range of growth factors and is widely used as a supplement in cell culture media (10,12–14,17,24). However, the use of serum poses a discrepancy in the context of fibroblast and keratinocyte co-culture. The presence of serum is required to support the proliferation of fibroblasts, which is in contrast to keratinocytes, which grow poorly or fail to attach and grow in the presence of serum, and are therefore cultivated in serum-free media (16,25–28). To overcome this, a large number of protocols for contact-based co-culture of fibroblasts and keratinocytes, start with growing confluent layers of fibroblasts in media supplemented with high concentrations of animal serum (up to 10%), after which keratinocytes are seeded on fibroblast layers in serum-free media (10,11,15,16,19). In doing so, the fibroblasts serve as a cell feeder system (feeder fibroblasts may be irradiated to provide additional factors that aid keratinocyte attachment) for proliferating keratinocytes, enabling their expansion (14).
Table 1
Comparison of different approaches for contact-based co-culture of dermal fibroblasts and epidermal keratinocytes
Co-culture Approaches | Serum Concentrations | Additional Growth Factors | Technical Ease | Ease of Media Formulation | Key Features | References |
Serum-based Approaches | | | | | | |
Keratinocytes layered on top of Fibroblasts (including cell feeder platforms) | ~ 5–10% FBS/FCS to grow fibroblast layers; certain protocols switch to serum-free media to layer keratinocytes | Present in commercial media or added individually as growth supplements | Utilizes standard cell culture techniques to layer cells; usually does not require irradiated cells or complex feeder systems | Media prepared from standard, commercially-available media components; supplements, if used, added individually | Supports co-culture of fibroblasts and keratinocytes (primary human cells and cell lines) using a layered approach | 5, 10, 16, 19, 24, 32, 33 |
Fibroblasts co-seeded with Keratinocytes | ~ 10% FBS/FCS | Additional growth factors, if used, added individually | Utilizes standard cell culture techniques to co-seed cells | Standard, commercially-available media components | Variable attachment of keratinocytes depending on serum and growth factors in media used | 11, 12, 14, 17, 24 |
Serum-free Approaches | | | | | | |
Keratinocytes layered on top of Fibroblasts | 0% | Additional growth factors, if used, added individually | Certain protocols lethally irradiate feeder layer of fibroblasts, on which keratinocytes are seeded | Media prepared from standard media components; supplements, if used, added individually | Supports expansion of keratinocytes; if irradiated feeder cells are used, unsuitable for cell migration and wound closure studies | 24, 32 |
Fibroblasts co-seeded with Keratinocytes | 0% | Additional growth factors, if used, added individually | Utilizes standard cell culture techniques to co-seed cells | Media prepared from standard media components; supplements, if used, added individually | Poor proliferation of HDFa cells and abnormal co-culture morphology without growth supplements | 12, 24 |
Our Reduced-Serum Approaches | | | | | | |
Fibroblasts layered on top of Keratinocytes | 1–2% FBS to form a confluent layer of fibroblasts and to seed keratinocytes over the fibroblast layer | Present in composite media or added via mixture of growth factors | Utilizes standard cell culture techniques to layer cells; does not use irradiated cells or complex feeder systems | Composite media prepared from cell-specific media or minimal media supplemented with mixture of cell-specific growth factors; precludes addition of individual growth supplements | Supports robust attachment, proliferation and cell morphology of HDFa and HaCaT cells in co-culture; cell migration and wound closure studies possible | This study |
Fibroblasts co-seeded with Keratinocytes | 1–2% FBS | Present in composite media or added via mixture of growth factors | Standard cell culture techniques; two cell types mixed and co-seeded | Composite media prepared from cell-specific culture media or minimal media supplemented with mixture of cell-specific growth factors; precludes addition of individual growth supplements | Supports robust attachment, proliferation and cell morphology of HDFa and HaCaT cells in co-culture; cell migration and wound closure studies possible | This study |
Declarations: |
While they have been successful, these approaches have certain limitations (Table 1). Several protocols employ high concentrations of animal serum (up to 10% FBS or FCS). The usage of animal serum in cell culture studies is associated with a range of well-known scientific, technical and ethical limitations, (29–31), and there has been a push to develop serum-free or serum-alternative approaches (31). Protocols that employ different media compositions for fibroblasts and keratinocytes require the two cell types to be cultivated in a layered fashion, with keratinocytes seeded on top of fibroblasts. While this broadly resembles the structure of the wound bed, to recapitulate the entire range of cell-to-cell interactions, the simultaneous attachment, proliferation, and contact of the two cell types is desirable (4,11,12,17).
Along these lines, a recent study compared co-culture results for human dermal fibroblasts (HDFa) and immortalized human epidermal keratinocytes (HaCaT) in the presence of serum and under serum-free conditions (12). HaCaT were either added soon after (~ 40 minutes) seeding HDFa (resembling co-seeding) or layered over confluent HDFa (after five days of HDFa growth). In both these approaches, the presence of serum was observed to variably influence the early attachment and proliferation of the two cell types. HaCaT added soon after seeding HDFa cells (~ 40 minutes), in the presence of 10% FBS (in Dulbecco’s modified Eagle’s medium; DMEM), displayed significantly impaired attachment to tissue culture plastic substrates; attachment of HaCaT cells was significantly higher under serum-free conditions. On the other hand, under serum-free conditions, HDFa displayed impaired proliferative capacity even after five days of culture (12). Notably, both media conditions, with serum and serum-free, did not contain additional cell-specific growth factors.
Other serum-free approaches employ the use of cell feeder platforms and synthetic media formulations (24,32–35). Cell feeder protocols typically lethally irradiate the feeder cells, usually fibroblasts, on which keratinocytes are seeded (Table 1). This results in growth-arrested fibroblasts, which are unsuitable for cell migration and wound closure studies. On the other hand, protocols using non-irradiated fibroblasts use media compositions that require a host of growth factors and supplements to be procured and added individually to the basal cell culture media (24).
Taken together, existing approaches for contact-based co-culture of dermal fibroblasts and epidermal keratinocytes are not only technically demanding and labor-intensive, but are also limited by differential effects of serum on the two cell types, need for complex cell feeder platforms, and material-intensive media formulations (Table 1).
Given this, it is necessary to develop methods that overcome these limitations, to successfully support the contact-based co-culture fibroblasts and keratinocytes, using approaches that can be leveraged to study cellular architecture, structure and functions of the wound bed.
We have formulated two reduced-serum based methods (1–2% FBS) to co-culture HDFa and HaCaT, in direct contact with each other. These approaches are simple, easy to formulate and use commercially-available media components. Using these reduced-serum approaches, fibroblasts and keratinocytes could be co-cultured by layering keratinocytes over confluent fibroblasts or by co-seeding the two cell types simultaneously. Both cell types demonstrated robust attachment, proliferation, and characteristic cell morphology when cultured together. Further, the co-cultured platforms under reduced-serum conditions could be used to study wound bed features, such as cell migration and wound closure. These approaches can be used and adapted for a range of wound studies, as well as skin, tissue and cell engineering applications, potentially reducing concerns with high-serum formulations.