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

Contact-based co-culture of fibroblasts and keratinocytes is important to study the structure and functions of the wound bed. Co-culture of these two cell types in direct contact with each other has been challenging, requiring high serum concentrations (up to 10%), feeder systems and a range of supplemental factors. These approaches are not only technically demanding, but also present scientific, cost and ethical limitations associated with high-serum concentrations. We have developed two reduced-serum approaches (1-2%) to support contact-based co-culture of human dermal fibroblasts (HDFa) and human epidermal keratinocytes (HaCaT). The two approaches include (1) Specialized cell culture media for each cell type mixed in a 1:1 ratio (KGM+FGM), and (2) Minimal media supplemented with cell-specific growth factors (MEM+GF). Co-culture could be successfully achieved by co-seeding (two cell types were introduced simultaneously), or in a layered fashion (keratinocytes seeded on top of confluent fibroblasts). With wound scratch assays, the co-cultured platforms could demonstrate cell proliferation, migration and wound closure. The reduced-serum conditions developed are simple, easy to formulate and adopt, and based on commonly-available media components. These contact-based co-culture approaches can be leveraged for wound and skin studies, and tissue bioengineering applications, potentially reducing concerns with high-serum formulations.


Introduction
In the proliferative phase of 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] (Figure 1A). Migration of keratinocytes and fibroblasts results in regions of direct contact and communication between the two cell types [4,5]. Interactions between these two cell types critically impact wound bed structure and functions, including cellular architecture, cell migration and wound closure [5,6]. 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. 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].
We have formulated two reduced-serum based approaches (1-2%) to co-culture fibroblasts and keratinocytes, in direct contact with each other. These approaches are simple, easy to formulate and use commonly-available media components. Using these reduced-serum approaches, fibroblasts and keratinocytes were co-cultured, by co-seeding the two cell types or by layering keratinocytes over confluent fibroblasts. 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.

Cell culture and maintenance:
Adult primary human dermal fibroblasts (HDFa) were obtained from PromoCell (Germany) and maintained in a of all-in-one ready-to-use Fibroblast Growth Media (FGM) (a proprietary formulation from Cell Applications, 116-500) containing 2% serum (FBS). Immortalized adult human keratinocytes (HaCaT) were a gift from Dr. Madhur Motwani (Linq Labs, Jehangir Clinical Development Centre, Pune, India) and were maintained in an all-in-one, ready-to-use Keratinocyte Serum-free Growth Media (KGM) (a proprietary formulation from Sigma, 131-500A). Cells were maintained at 37°C and in a 5% CO₂ humidified incubator. Co-culture was done in T25 flasks (Tarsons) and 6-well tissue culture treated plates (Thermo Scientific), with total media volumes of 5mL and 2mL respectively. Cell counting was done with a hemocytometer. Cells were seeded at a density of ~10⁵ cells/mL, unless stated otherwise. Imaging was performed at 10X magnification with a Magnus INVI microscope equipped with a Magcam DC5 camera. For all experiments in MEM, Penicillin-Streptomycin (Gibco) was added to media (with or without serum) in a final concentration 1X.

Reduced-serum media formulations for co-culture:
To co-culture HDFa and HaCaT in the presence of reduced-serum, two media formulations were developed. In one of them, equal parts of FGM (containing 2% serum) and KGM were mixed (in a 1:1 ratio), resulting in composite media with an effective concentration of 1% serum (FGM+KGM). In the second formulation, Minimum Essential Media (MEM) containing Earle's salts, 2mM L-Glutamine, 1mM Sodium pyruvate, Non-Essential Amino Acids (NEAA) and 1.5gms per litre sodium bicarbonate (MEM, Himedia, AL047S), was supplemented with fibroblast growth factor (FGF) (Gibco, S00310) and keratinocyte growth factor (KGF) (Gibco, S0015) at concentrations of 1X each (MEM+GF). FGF contains fetal bovine serum, hydrocortisone, human epidermal growth factor, basic fibroblast growth factor and heparin. When FGF is added to the media at a concentration of 1X, the resulting media contains 2% FBS. KGF contains bovine pituitary extract, recombinant human insulinlike growth factor-I, hydrocortisone, bovine transferrin and human epidermal growth factor, and is serum-free.
Co-culture using co-seeding and layering techniques: HDFa and HaCaT cells were co-cultured in reduced-serum conditions, using two approaches using (1) a co-seeding technique, in which the two cell types were introduced simultaneously and (2) in a layered fashion, with keratinocytes seeded on top of a confluent layer of fibroblasts.
For the co-seeding approach, HDFa and HaCaT cells were counted and seeded in 1:1 ratio into both T25 flasks and 6-well plates. Both reduced-serum formulations, FGM+KGM and MEM+GF, were employed. The T25 flasks were imaged at 10X every 24 hours for 5 days. The 6-well plates were imaged at 10X every 24 hours intervals till 70-80% confluent.
For the layering approach in FGM+KGM media, HDFa cells were counted and seeded in T25 flasks in FGM (2% serum), incubated at 37°C and grown to 90% confluency. When confluent, the FGM was removed and replaced with FGM+KGM (in a 1:1 ratio; 1% serum). HaCaT cells were counted and seeded on the confluent layer of fibroblasts. Flasks were imaged at 10X every 24 hours for 5 days.
For the layering technique in MEM+GF, HDFa cells were counted and seeded in T25 flasks in MEM supplemented with 1X FGF, incubated at 37°C and grown to 90% confluency. When confluent, the media was replaced with MEM containing both FGF and KGF (1X each; 2% serum). HaCaT cells were counted and seeded on the confluent layer of fibroblasts. Flasks were imaged at 10X every 24 hours for 5 days. Similarly, the layering approach is also performed in 6-well plates for both the media conditions ( Figure S2A).

Wound scratch assay on co-cultured platforms:
HDFa and HaCaT were counted and seeded in 6-well tissue culture treated plates in reduced-serum conditions, with MEM + GF, using the co-seeding or layering approach. When the co-cultured cells reached 90% confluency, the media was replaced with fresh media, and a scratch was made across each well using a sterile 200uL tip. Following this, the wells were imaged at 10X at 0, 4, 24, 48 and 72 hours (three images were captured per scratch at different regions). The images were analyzed in ImageJ [14] by drawing a freehand outline of the scratch and using ImageJ tools to calculate the area within the outlined region (Suppl. Fig. 2). This was done for three images per well for three replicates for each condition, and an average wound area was calculated.

Reduced-serum conditions developed using commonly-available media components
Two reduced-serum approaches were formulated using commonly-available media components concentration each, referred to as MEM+GF. 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).

Reduced-serum conditions support the co-culture of HDFa and HaCaT cells using the coseeding technique
For the co-seeding approach, human dermal fibroblasts (HDFa) and adult human keratinocytes

Reduced-serum conditions support the co-culture of HDFa and HaCaT cells using the layering technique
For the layering approach, HDFa cells were grown to 90% confluency, following which HaCaT cells were counted (~10⁵ cells/mL) and seeded on layers of confluent HDFa. Starting from Day 1, HaCaT cells were observed to attach on the HDFa layers under both reduced-serum conditions. As in the coseeding approach, HaCaT cells proceeded to form distinct clusters on and between sheaths of HDFa cells starting from Day 1 (MEM+GF) or Day 2 (FGM+KGM), which continued to proliferate and increase in size ( Figure 2B).

Reduced-serum approaches developed support characteristic HDFa and HaCaT morphologies under co-culture conditions
In co-culture under reduced-serum conditions, HaCaT cells displayed typical polygonal cell clusters (colonies), with mosaic, cobblestone cell morphology, and HDFa cells were observed to form characteristic sheaths of spindle-shaped cells (Figure 2 and Figure S1A). Notably, the reduced-serum conditions developed included cell-specific growth factors (as components of specialized cell media or added into basal media), which, along with optimum serum concentrations, was observed to be critical to establish co-culture and characteristic cell morphology ( Figure S1B). As seen in Suppl.

The co-cultured platform, under reduced-serum conditions, enables the study of cell migration and wound closure
HDFa and HaCaT cells were co-cultured in tissue culture treated 6-well plates ( Figure S2A) under reduced-serum conditions (with co-seeding and layering approaches), following which a scratch was applied across the co-cultured cells. As seen in Figure 3A Figure   3B). Similar to that observed with the co-seeding approach, fibroblasts migrated into the wound gap, while keratinocyte clusters were seen to increase in size and fill the wound area from the periphery.
The wound area was determined by drawing a freehand outline of the scratch and using ImageJ tools to calculate the area within the outlined region ( Figure S2B). This was done for three images per well for three replicates for each condition, and an average wound area was calculated.

Discussion
Contact-based co-culture of human dermal fibroblasts and epidermal keratinocytes is important for a range of wound and skin studies. Previous approaches to establish co-culture of fibroblasts and keratinocytes in direct contact with each other are often tedious and labor-intensive, and notably employ high-serum concentrations (often up to 10% FBS). Animal serum, for example fetal bovine serum (FBS), is widely-employed in cell culture studies, and possesses a range of active substances, known to support the proliferation and morphology of a variety of cell types. However, its usage in high concentrations is associated with several limitations [11][12][13], which are particularly relevant in the context of co-culturing fibroblasts and keratinocytes. The attachment, proliferation and cell morphology of fibroblasts and keratinocytes varies depending on the concentration of serum in the media and presence of specific growth factors. For example, keratinocytes (HaCaT) co-cultured with fibroblasts (HDFa) in the presence of 10% fetal bovine serum, displayed poor attachment to tissue culture plastic substrates; whereas when co-cultured in the complete absence of serum, HDFa cells demonstrated impaired proliferative capacity [7,9,15]. Further, in serum-free media, both cell types failed to form characteristic cellular arrangements. Given this, there is the need to develop approaches that successfully support the co-culture of fibroblasts and keratinocytes at concentrations of serum and growth factors, that not only overcome the differential effects of serum on the two cell types, but are also easy to formulate and adopt in experimental practice.
The reduced-serum approaches developed in this study, support the co-culture of fibroblasts and keratinocytes, using both co-seeding and layering approaches. These co-cultured systems support the proliferation and cell morphology of both cell types, and enable the study of wound bed functions such as cell migration and wound closure. The formulations developed result in effective concentrations of 1% FBS in KGM+FGM, and 2% in MEM+GF, representing a 5-10 fold reduction in serum concentration as compared with previous studies, using up to 10% serum [7,9,15]. Further, these approaches are easy to formulate and use commonly-available media components, making them relatively simple to adopt and use. Finally, reduced-serum approaches align with the push to reduce the scientific, technical and ethical limitations [11] associated with the use of animal serum in cell culture. These limitations include substantial batch-to-batch variation, presence of uncharacterized factors, and potential risk of microbial contamination, likely to influence experimental outcome and reproductivity. Further, procedures to harvest serum from animal sources are poorly regulated and standardized, posing major ethical and moral concerns. These limitations can be potentially alleviated with the development and application of reduced-serum formulations.
To the best of our knowledge, there is no previous work reporting the contact-based co-culture of fibroblasts and keratinocytes under reduced-serum conditions (1-2%) with cell-specific growth factors, as reported in this study. These approaches can be leveraged and adapted for a range of wound studies, as well as skin, tissue and cell engineering applications, potentially reducing concerns with high-serum formulations.

Conclusions
We have developed two reduced-serum based approaches that support the co-culture of fibroblast and keratinocytes in direct contact with each other. These co-cultured systems support the robust attachment, proliferation and cell morphology of both cell types, using co-seeding and layering techniques, and enable the study of wound bed functions such as cell migration and wound closure. Further, these approaches are simple to formulate, easy to adopt, use widely-available media components, and potentially reduce concerns associated with high concentrations of animal serum. Taken together, these protocols lend themselves well for co-culture of these two cell types for wound and skin studies, tissue bioengineering and cell therapy applications.