Wounds such as those caused by burns, ulcers and physical impact result in a breach of the human skin barrier, subsequently exposing the underlying tissue, which can lead to opportunistic microbial invasion and establishment of infection. The body’s immediate response to skin damage is a complex and dynamic biological process known as wound healing that is driven by the spatially and temporally coordinated action of various cell types and extracellular factors (Dunnill et al., 2017). However, wounds that are either highly extensive (such as burns) or complex and non-healing (such as diabetic ulcers) greatly curtail the effectiveness of clinical wound management, leading to extended patient care thus resulting in enormous financial burden for healthcare providers. For the National Health Service (NHS) in the United Kingdom, the annual financial burden of care for patients with wounds and associated comorbidities in 2012/13 was £5.3 billion, with a 135% increase in the cost of care for patients with unhealed wounds compared to healed wounds (Guest et al., 2017); the cost of wound management is now estimated to have risen to £8.3 billion, of which £2.7 billion and £5.6 billion were associated with managing healed and chronic wounds, respectively (Guest et al., 2020).

Historically, treatment of extensive wounds has utilised split-thickness skin grafting (STSG), whereby an autologous skin graft is taken from the patient and placed onto the wound site, allowing for the donor site to heal. This method of intervention however is relatively outdated and, in some cases, poses caveats including inefficiency in healing, aesthetic displeasure and lengthened patient hospital stays due to large amounts of donor site skin used (Bayat et al., 2003; King et al., 2014; Guest et al., 2017). As a result, there is an urgent clinical need for producing novel, alternative approaches to achieve rapid and efficient recovery in wound care and improvement in the overall wellbeing of patients. Examples of alternative approaches include epidermal grafting which may deliver similar healing rates to STSG, yet only the epidermal layer of skin is harvested, leading to reduced utilisation of donor site resources (Kanapathy et al., 2016; Kanapathy et al., 2021). Another approach involves the use of bioengineered tissue (in vitro or 3D bio-printed) whether epidermal, dermal or composite, permanent or temporary skin replacements, these skin substitutes primarily function as barriers for prevention of fluid loss and microbial contamination (Vig et al., 2017; Sörgel et al., 2023). However, bioengineered artificial skin grafts are not yet fully optimized, and are currently limited in their clinical application due to financial and time-consuming constraints in the production process (Przekora, 2020; Sörgel et al., 2023).

Other approaches encompass isolation and expansion of cells (keratinocytes and fibroblasts), to significant quantities and subsequently they can be utilised in various procedures such as allogeneic cell therapy, whereby cells are preserved until required for direct application, or introduced to constructs such as scaffolds (Kirsner et al., 2012; Chen et al., 2023) However, use of allogeneic cells is limited due to immune rejection, and patients may require immunosuppressive drugs in concomitance to the treatment (Jorgensen et al., 2023).

Despite these promising developments, STSG still represents the main method of choice in wound care, because these novel alternative strategies have been unable to contemporaneously offer improvements in cost-effectiveness, ease of practical use (whereby a surgeon or nurse could operate the technology), and/or show similar or considerable effectiveness in accelerating the healing response compared to STSG. One promising type of strategy that has the potential to overcome such obstacles is through a cell-based, “point-of-care” therapy which involves use of autologous (rather than allogeneic) skin cells (Zhao et al., 2016). The principle of this approach involves enzyme-based solutions to isolate viable and proliferative cells from a patient’s own skin in situ, which can then be immediately applied to the wound area (Wood et al., 2012; Holmes IV et al., 2018; Manning et al., 2019). An attractive advantage of autologous cell-based therapies is that the formation of an autologous cell suspension (ACS) requires a much smaller amount of donor site skin. Unlike using STSG, which requires donor to wound site ratios of 1:1 or 1:2 (whether meshed or not), ACS application may require remarkably less donor skin, and in some cases it has been suggested that even an 1:80 ratio might be effective (Holmes IV et al., 2018). However, although the concept of autologous cell-based therapy is highly promising, it has not shown adequate clinical success, and the associated cost of currently available devices for ACS isolation remains extremely prohibitive.

To overcome the practical and cost-associated limitations of the currently available laborious methods and expensive devices for preparation of ACS, we have recently established a novel method for rapid, enzymatic disaggregation of human skin cells and their isolation using a patented procedure that requires no specialist laboratory instrumentation and is performed at room temperature. Here, we report for the first time the characterisation of the types of cells isolated and their viability, we provide evidence that the isolated ACS contains epidermal stem cells, and we define the biological properties of the keratinocyte population, which include their ability to secrete positive mediators of wound healing as well as their capacity to modulate myofibroblast cytodifferentiation.

The process of skin re-epithelialisation during wound healing is facilitated by rapid proliferation and migration of keratinocytes; failure or deceleration of this step in the healing process is associated with delayed tissue repair, infection and/or chronic wound development. There is increasing evidence that autologous cell suspension-based therapies represent a highly promising approach (Zhao et al., 2016) for burns and various type of chronic wounds (Shukla et al., 2010; Hu et al., 2015; Hayes et al., 2020) and with increasing clinical success (Holmes IV et al., 2018; Kowal et al., 2019). It has been postulated that ACS-associated cells successfully integrate (“take”) into a wound site (Sood et al., 2015; Domaszewska-Szostek et al., 2021) which accelerates re-epithelisation and wound healing, reduce the risk of infection and suppresses discomfort (pain). However, despite the great promise of the approach, the existing technologies for the application of ACS (in the form of ‘spray-on’ skin via a syringe-like applicator or a more sophisticated ‘spraying gun’) have not always achieved the desired clinical promise due to a number of limitations such as high cost versus clinical benefit (Manning et al., 2022) whilst obtaining optimal cell populations often requires electrical and/or laboratory instrumentation (Esteban-Vives et al., 2015; Esteban-Vives et al., 2016). Therefore, in light of this clinical promise, it is vital that improved approaches address these limitations whilst ensuring that the established ACS cell populations have all the desired biological properties to enhance wound closure as well as healing ‘quality’, i.e., to restore pigmentation and to ideally suppress scarring.

Our aim is to transform existing ACS technologies for rapid preparation of autologous cell populations in solutions that contain highly-viable cell populations, These can be applied directly and immediately (without culturing) upon isolation in the operating theatre in order to accelerate wound healing. Here we have provided evidence that our novel method permits the rapid establishment of autologous cell suspensions at room temperature and without the use of specialist instrumentation, which: a) include viable keratinocytes, fibroblasts and skin pigmentation-associated melanocytes as well as epidermal stem cell populations, whilst b) the ACS-derived cells possess wound-beneficial biological properties, such as the ability to release positive mediators of wound healing, ability to enhance human keratinocyte proliferation, whilst suppressing the overactivation of myofibroblasts.

Our study has confirmed that the ACS isolates comprise highly viable population of keratinocytes, fibroblasts and melanocytes. Rheinwald and Green first originally described methods for isolation and cultivation of viable keratinocytes from skin, whilst demonstrating the importance of fibroblast ‘feeder’ layers to support keratinocyte growth (Rheinwald and Green, 1975). These feeders aim to support growth by secreting growth factors to the keratinocytes, whilst producing ECM components to facilitate keratinocyte adherence to the culture substratum (Tracy et al., 2016). Using a Collagen IV-coated substratum, we found that this wound-bed mimic was adequate to support rapid recovery and proliferation of keratinocytes in the absence of feeders, which confirmed the quality of the isolates (i.e., high level of ACS cell viability). It is also noted that our filtration method for isolation of skin sub-populations permitted the removal of excess extracellular matrix (ECM) proteins/fragments which was important in the initial attachment of cells (particularly keratinocytes). Critically, without removal of such ECM fragments we observed that cell attachment to the collagen substratum was significantly compromised, which notably curtailed adherence and thus cell recovery (data not shown).

A previous report on the establishment of autologous cell suspensions has provided evidence for recovery of 1.7 × 10⁶ cells per cm² of human skin with ∼75% viability (Wood et al., 2012); notably, the report involved use of technology that utilises specialist electrical instrumentation and requires incubation (heating) of the skin specimens. Here, we report cell recovery of ∼5 × 10⁵ cells per cm² tissue with an ∼85% viability at the point of isolation. Importantly, however, we note that our cell yields and observations on cell behaviour (particularly speed of reaching culture confluency) are in complete agreement with the previously reported, optimal methodologies for establishment of keratinocytes, which represent the ‘gold-standard’ method for human skin processing and keratinocyte isolation. Yet, such optimal procedures involve overnight dispase treatment and subsequent trypsin-based isolation procedures, which requires use of cell culture facilities and specialist instrumentation (Aasen and Izpisua Belmonte, 2010).

Stratum corneum is the outermost epidermal sub-layer represented by a keratinized barrier that is formed by terminally differentiated keratinocytes, which are continually replenished by epidermal stem cells (EpSCs) and transit amplifying cells (TA) residing in the basal layer of the epidermis. EpSCs are hypothesised to be “intrinsically ageing resistant” (Stern and Bickenbach, 2007; Giangreco et al., 2008) and are important not only in adult skin homeostasis, but also in restoring the epidermal barrier after injury (Blanpain and Fuchs, 2006). Epidermal cells in the supra-basal layer have also been shown to de-differentiate and revert to stem cells upon wounding, further supporting the role of the stem cell phenotype in tissue repair (Fu et al., 2001; Mannik et al., 2010) thus substantial skin repair is inextricably linked with the presence of these EpSCs. However, to date, no definite singular marker for EpSCs has been identified and a combination of markers provides a sub-population that shows stem-like properties and functionality. This contrasts to the hair follicle, whereby proteins such as CD200 are used to specifically mark stem cells of the bulge (Purba et al., 2017). Postulations for putative EpSC markers are ongoing, with combinations of cell surface expression of α6-integrin and CD71 being the most extensively characterised. Integrin α6 (also known as CD49), is a cell-surface protein exclusively expressed on the basal cells of the epidermis, which marks both stem and TA cells (Webb et al., 2004). CD71 (transferrin receptor) is highly expressed in actively cycling cells, yet it is expressed at low levels in long-term proliferating neonatal foreskin cells as well as cells in the human hair follicle bulge region (Tani et al., 2000; Webb et al., 2004). It has been reported that the α6^high/CD71^low expression pattern demarcates cells with stem cell-like properties, while α6^high/CD71^high represent TA cells. Importantly, cells with the α6^high/CD71^low phenotype exhibit more potent epidermal renewal capacity (Webb et al., 2004; Metral et al., 2017). In support of these observations, the α6^high/CD71^low phenotype also seems to define a stem cell population in other types of epithelia, such as oesophageal and corneal (Croagh et al., 2007; Hayashi et al., 2008). Here, we have shown that the ACS isolates comprise cells with the α6^high/CD71^low expression profile and a population % that is in accordance with previous observations for the type of skin we used, in our studies mainly abdominal skin samples (Metral et al., 2017). Notably, we observed the same α6^high/CD71^low expression profile whether ACS cells were immuno-labelled directly after isolation or following initial overnight attachment and recovery (not shown). Although we did not specifically isolate this cell sub-population for further assessment of self-renewal capacity and clonogenicity, we suggest that the presence of these cells would be supportive of the high proliferative capacity of our ACS-derived cells based on observations by Webb et al. (2004) and Metral et al. (2017) that the CD49/CD71 EpSC sub-population drives epidermal regeneration in vitro and in vivo; thus the presence of such cells confirms the ‘quality’ of our ACS isolation procedure.

Moreover, as the aim is to use ACS isolates from a patient’s own skin in situ immediately to accelerate wound healing, the presence of the EpSC population could be critical in the ability of ACS to enhance wound healing and it is significant, in line with the strong promise of stem cell-based therapies for cutaneous wound healing (Kosaric et al., 2019).

Our study has demonstrated that immediately upon establishment and attachment to a wound bed mimic, the ACS cell populations exhibited biological properties that can enhance the wound response by providing beneficial extracellular mediators to the surrounding wound bed. We found that the ACS secretome contains a large panel of growth factors including EGF and FGF, which are important for keratinocyte proliferation and motility (Werner and Grose, 2003; Park et al., 2018) as well as containing various cytokines and chemokines which are well-characterised promoters of keratinocyte proliferation and motility (Maas-Szabowski et al., 1999; Spiekstra et al., 2007; Kroeze et al., 2012), with pro-inflammatory cytokine IL-1α prominently over-expressed in the ACS secretome. We also detected more recently established positive mediators which are not attributed to viable cells but can be released from perishing (non-viable) cells via processing and isolation procedures to establish ACS from skin. Damage-associated molecular pattern (DAMP) proteins, and HMGB1 and HSP90α in particular, are molecules released from dying cells that not only promote innate immune pathways but are also associated with promoting ‘wound-edge’ keratinocyte proliferation and motility (Li et al., 2007; Ranzato et al., 2009; Li et al., 2012; Bhatia et al., 2016; Bhatia et al., 2018). We observed a steady and gradual rise of HMGB1 levels during culture, while HSP90α displayed its highest concentration on the first day of culture and its levels remained sustained throughout. The prompt detection (<24 h of culture) suggests that administered ACS may have an immediate effect on ‘wound edge’ keratinocytes whilst also releasing beneficial soluble factors that can enhance wound healing.

We have also provided evidence that at least some of the components of the ACS secretome are mitogenic factors, since conditioned medium (neat or diluted) from cultured ACS isolates enhanced the proliferation of both immortalised (HaCaTa) and, more importantly, primary keratinocyte (NHEK) cultures. Interestingly, these important findings are in contrast to observations from a previous report, where no such positive effects in proliferation was observed for HaCaT cell cultures treated with CM from ACS (Wood et al., 2012). We believe that the lack of enhancement of cell proliferation observed by Wood et al. would be attributable to the use of the original HaCaT cell line. As such cells are cultured in serum-containing medium, the presence of serum could have significantly ‘masked’ any positive effects by the diluted ACS CM.

Human dermal fibroblasts (HDFs) play important roles during would healing, including the responsibility for breaking down the blood clot during healing and producing new ECM structures to support other cells involved in tissue repair. Furthermore, HDFs can differentiate to the phenotypically larger myofibroblasts which express smooth muscle actin (α-SMA). This protein is central in the contractile properties in myofibroblasts to facilitate the mechanics of physically pulling the wound edges together. Typically, when tissue repair is complete, myofibroblasts undergo apoptosis, however if their presence is inappropriately sustained when dysfunctional tissue repair occurs, subsequently causing excess contraction and fibrosis (Penn et al., 2012; Darby et al., 2014). Thus, extensive myofibroblast activity is detrimental to optimal wound healing, leading to scarring and overall poorer healing aesthetics for patients. Furthermore, TGF-beta is abnormally expressed in instances of delayed wound healing, resulting in higher myofibroblast activity, inhibition of fibroblast proliferation, excessive laying of extracellular matrix and higher tension during contraction (Goldberg et al., 2002). It was therefore of interest to investigate whether the application of ACS CM on a wound could affect myofibroblast homeostasis. Using a well-characterised in vitro model of HDF differentiation to myofibroblasts, we have provided evidence that our ACS CM regulated the phenotype of HDFs by suppressing the process of cytodifferentiation induced by low density/TGF-β treatment, evident by the reduction in α-SMA expression. Although it remains unclear which specific paracrine factors may have mediated these suppressory effects, clearly the ACS secretome comprises a substantial number of soluble factors, of particular note being EGF and FGF. Previous studies have reported that EGF activity can negatively regulate myofibroblast differentiation via MEK/ERK- and JNK-dependent signalling (Kimura et al., 2013). More importantly, there is extensive evidence for a central role for FGF signalling in curtailing TGF-β induced myofibroblast differentiation, as FGF-2 (known as basic FGF) attenuates scarring during cutaneous wound healing (Spyrou and Naylor, 2002) by suppressing myofibroblast establishment and inducing their apoptosis (Akasaka et al., 2004; Dolivo et al., 2017). The suppressory effects of FGF signalling on TGF-β mediated transition of fibroblasts to myofibroblasts is also supported in the context of disease, such as cancer (Akatsu et al., 2019). Notably, our ACS secretome studies provided evidence for marked increase in FGF-2/FGF-basic expression by Day 3 post culture and, strikingly, it was ACS CM from Day 3 cultures which was used in our experiments (whilst Day 1 culture ACS CM did not show such effects–data not shown). Thus, it is tempting to speculate that suppression of myofibroblast differentiation by our ACS isolates is FGF-2 mediated, however functional FGF-2 inactivation studies would be required to confirm this scenario. Yet, irrespective of the soluble effector mediating these effects, our observations have provided strong evidence that in addition to enhancing keratinocyte proliferation, the ACS-derived cells can attenuate myofibroblast overactivation thus potentially enhancing not only the speed but also the ‘quality’ of wound healing.

In conclusion, based on the recent establishment of our novel methodology for the generation of ACS from split-thickness skin grafts, our study has systematically investigated the sub-populations of cells in the ACS isolates and their biological properties, which collectively are expected to be beneficial upon application to a wound (Figure 6). We demonstrate several important wound-beneficial features including presence of vital cell populations as well as positive modulation of critical steps in wound response (keratinocyte proliferation and suppression of myofibroblasts), and therefore our findings merit further investigation by in vivo studies to demonstrate wound benefit. Nevertheless, our work has provided several lines of evidence that the ACS isolates established not only comprise highly viable cell populations that can physically support wound healing but also possess biological properties that have the potential to enhance wound healing, and thus to compensate for the deficiency in essential elements (both viable cells and soluble factors) that are absent in non-healing wounds.