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[Full text] Skin models for the testing of transdermal drugs | CPAA
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Soft Matter 6, — The printing stage is then completed using different methods such as inkjet printing [ 33 , 35 , 36 ]. Inkjet printing uses bioink for the process of bioprinting and may include the use of cells as the bioink [ 37 , 38 ]. As discussed previously, the results obtained from in vitro 2D monolayer cell culture models have not translated to in vivo successes [ 7 ]. Thus, alternative methods for testing dermatological products have been sought out.
Skin cell cultures that are 3D have the advantages of containing a stratum corneum and thus the ability of testing pharmaceutical products on the stratum corneum as a skin barrier [ 40 ]. The main disadvantage to the use of 3D skin cell cultures however is the cost associated with developing these cultures [ 40 ]. The different types of 3D skin cell cultures that will be discussed in detail are histocultures, human skin equivalents, on-chip skin cultures, and pigmented cell cultures. A discussion on bioprinting of skin constructs is also included.
Histocultures are cultures of intact tissues that were developed to better mimic in vivo skin responses [ 42 ]. The process of hair growth is an example of the ability of histocultures to mimic in vivo processes, as this process occurs in histocultures of skin cells and allows for the testing of pharmaceutical products aimed at inhibiting or improving hair growth [ 42 ].
The process of histocultures involves growing skin tissues on a growth medium on its own or with the support of collagen [ 42 , 43 ]. Research has shown that both epidermal and dermal cells as well as hair follicles maintain their functions and physiology in skin histocultures [ 44 ]. Histocultures have thus successfully been used as in vitro models for testing dermatological products, particularly with respect to toxicity screening [ 42 , 44 ]. In order to test pharmaceutical products on the skin in vitro and provide suitable skin replacement options for patients with various skin conditions, such as burn victims, HSEs were created [ 45 ].
HSEs are 3D cell culture models created from various human skin cells and materials that mimic the extracellular matrix [ 45 ] and are created as either epidermal equivalents, dermal equivalents or skin equivalents consisting of both layers [ 8 , 45 ]. Keratinocytes are then incubated for 2 days following their transfer onto a de-epidermized dermal equivalent and then placed at air-liquid interface for the development of full thickness human skin equivalent [ 46 ]. As these skin substitutes are derived of only the epidermal layer and primarily keratinocytes, it limits their use for testing of products related to particular types of skin conditions that involve the immune system, including testing of products for wound healing [ 47 ].
Full thickness models consisting of both the epidermal and dermal layers are thus beneficial [ 8 ]. Microfabricated systems, also referred to as cells on chips, are a type of 3D cell culture that uses microtechnology to provide cells with the required growth environment that can be easily controlled [ 30 ]. On-chip culturing also uses microfluidics for providing nutrients [ 30 ]. This model provides an opportunity for dermatological medication testing on a microfabricated cell system as growth and differentiation of skin cells were made possible through this model [ 50 ].
The comparison revealed that the static chip was not the ideal method as differentiation of skin did not occur and the epidermis was not attached by the end of 1 week [ 52 ]. It is thought that this is a result of lack of flow or perfusion in the static chip and thus insufficient flow of nutrients for the growth of the cells [ 52 ].
Thus an advantage to using microfabricated skin cell cultures vs. Using a dermal equivalent with a steel ring on top, seeding of human keratinocytes and melanocytes was completed [ 53 ]. The culture developed a monolayer after a period of 1 week in medium after which it was exposed to air for another week to allow differentiation of keratinocytes [ 53 ]. This 3D model allows for a better understanding of the interactions between melanocytes, keratinocytes, and fibroblasts [ 53 ] and also allows for a pigmented human skin model that could potentially be used for testing of pharmaceutical products on pigmented skin.
The process of bioprinting explained above is still the same process followed for bioprinting of skin constructs. An important consideration, however, in the pre-processing stage, is that the imaging equipment used ideally should be able to differentiate skin color [ 34 ]. Also, with respect to cell selection, keratinocytes are the primary cell types used for bioprinting of skin cells [ 34 ]. The advantages to using bioprinting of skin constructs includes greater accuracy in placement of cells and extracellular matrix as well as having the potential of imbedding vasculature in the skin construct as bioprinting of vasculature is also possible [ 54 ].
Skin constructs made through bioprinting are also considered to have great plasticity [ 54 ]. Skin bioprinting may also be used for developing 3D models for drug testing, such as diseased skin models, and are believed to provide more uniform models compared to manually developed skin models [ 55 ]. The main disadvantage of bioprinting for skin constructs is the high cost associated with its use [ 54 ].
Wound healing is a physiological process that consists of four phases: hemostasis, inflammation, proliferation, and remodeling [ 56 ]. In the first phase, after a wound injury, hemostasis, platelets are activated and migrated to the site of injury [ 57 ]. The second phase, inflammation, begins about 1 day postinjury and inflammatory mediators such as histamine are released, providing the typical traits of inflammation such as heat and swelling [ 57 ].
Proliferation is the phase in which granulation tissue forms at the site of injury and after which re-epithelization occurs [ 58 ]. The normal wound healing process could be affected, however, leading to chronic ulcers or excessive wound healing resulting in hypertrophic scars [ 59 ]. For this reason, topical agents to improve wound healing or reduce scarring may be of interest and as such in vitro testing models for wound healing will be discussed.
Both 2D and 3D skin cell cultures are available as wound healing models [ 60 ]. Cells in 2D monolayer cultures are thought to adhere to the flat environments, such as a Petri dish, in which they are cultured and will therefore migrate to areas of free space within the dish, an activity thought to mimic in vivo migration involved in cell differentiation [ 62 ]. One method of mechanical introduction of a wound to a 2D cell culture is through the scratch assay that utilizes materials such as pipette tips or needles to introduce a wound or scratch into the monolayer cell culture [ 63 , 64 ].
Images of the wound are taken within set time frames to assess the migration of cells [ 63 ]. Typically, however, it is difficult to ensure wounds that are equal in size using this method [ 63 ], and thus for this reason, testing of pharmaceutical products on these types of cultures are not ideal. Histocultures are cultures of intact tissues, consisting of more than one type of skin cell, such as neutrophils and other cells involved in wound healing, and are thus able to better mimic in vivo skin responses of wound healing [ 42 , 43 ].
HSEs, on the other hand, are 3D cell culture models created from various human skin cells and materials that mimic the extracellular matrix [ 45 ] and are created as either epidermal equivalents, dermal equivalents or skin equivalents consisting of both layers [ 8 , 45 ]. Some examples of 3D wound healing skin cell cultures and HSEs are described below.
As angiogenesis is involved in the process of wound healing [ 66 ], this model is an excellent example of improved 3D wound healing cell culturing protocols to more closely mimic in vivo wound healing processes. This model allows for the immersion of biopsies in Williams E culture media with the epidermal layer of the skin uncovered and uses whole tissue biopsies in well cell plates with transwell inserts [ 67 ]. This model allows application of topical medications to the uncovered epidermal layer and is thus useful for testing of pharmaceutical products, including those used for transdermal drug delivery [ 67 ].
The skin substitutes derived of only the epidermal layer and primarily keratinocytes, however, are limited in their use for testing of products related to particular types of skin conditions that involve the immune system, including the testing of products for wound healing [ 47 ]. This kit can be purchased with a wound healing assay kit [ 49 ] and is thus a commercially available 3D model for testing of dermatological products aimed at wound healing.
It is thus evident from the examples provided in this section that 3D wound healing models more closely mimic in vivo wound healing processes and thus may be a better choice for testing of pharmaceutical products aimed at wound healing. Wound healing models are also commercially available which may improve the use of in vitro models as a replacement for animal testing. As an inflammatory skin condition that involves the immune system, psoriasis has the characteristic appearance of silver scales that arise from increased proliferation of the keratinocytes in the epidermal layer [ 68 , 69 ].
Psoriasis is typically treated with topical steroid medications or with vitamin D analogue medications such as calcipotriol as well as with moisturizing agents [ 68 ]. The treatment of psoriasis also includes systemic medications that suppress the immune system such as methotrexate and cyclosporine as well as biologic drugs [ 68 , 70 ].
Psoriasis has been linked to various mental health illnesses such as anxiety and depression which are thought to result from having a chronic visible skin condition [ 71 ], and therefore the need for developing new pharmaceutical products and testing of these products is evident. As psoriasis is primarily treated with topical medications [ 70 ], having in vitro cell cultures or models for testing the safety and efficacy of these topical medications is essential. Skin cell cultures that are 2D for dermatological conditions such as psoriasis and other autoimmune disorders exist [ 39 ].
As a result, alternative methods such as adding cytokines to normal human keratinocytes to induce psoriatic features were developed [ 73 ]. As stated previously, however, testing of medications on 2D cell culture models does not always translate to in vivo responses [ 7 ], and thus, 3D models to better mimic in vivo responses for testing of topical medications for psoriasis have been developed. For skin substitutes, ascorbic acid was used to culture the fibroblasts which formed dermal sheets that were altered to create a dermal layer onto which keratinocytes were then seeded to form an epidermal layer [ 75 ].
This method is the self-assembly method as the fibroblasts release their own extracellular matrix to maintain their growth [ 75 ]. This was compared to healthy skin substitutes and revealed that the psoriatic skin substitute had a greater permeability response to the three compounds [ 76 ]. For safety and efficacy testing of medications for psoriasis, commercially available 3D in vitro models also exist.
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- Cell Culture Models of Biological Barriers: In vitro Test Systems for Drug Absorption and Delivery.
Skin is an intricate organ of the human body and serves many essential physiological functions for human survival such as fluid homeostasis, thermoregulation, immune defence, and sensory detection. It forms an efficient physical barrier to protect the body against environmental pathogens, toxic chemicals, mechanical disturbances, and UV radiation 1. Due to its accessibility and large surface contact area, skin is considered as a suitable and vital route for administration of drugs or application of cosmetic products 2 , 3.
Testing of these substances on the skin is thus of crucial importance to assess the dosing and therapeutic efficacy, to identify the potential adverse skin reactions and mode of action, and to analyze human environmental risks 4. Currently, animal models are extensively used for such drug testing, but they are usually lacking availability, highly time-consuming and costly, ethically questionable and may not represent the physiology, immunity and metabolism of the human skin, resulting in a limited ability to extrapolate to human conditions 5.
Thus, human skin equivalents HSEs for drug testing using developed in vitro skin models are considered valuable tools for studying the molecular basis of cellular responses in skin physiology and pathology 6 , 7. Conventional two-dimensional 2D culture models have involved cultures of keratinocytes or co-culture of keratinocytes with immune cells and dermal fibroblasts on petri-dishes or microtiter plates 1. These models are well-established and straightforward to use; however, they fail to reconstitute the complex three-dimensional 3D cell-cell and cell-matrix interactions found in the body, limiting their accuracy in predicting the complicated effect of drug metabolism on the actual skin.
To tackle these limitations, development of 3D skin models with cells cultured in extracellular matrix ECM -like materials e. Typically, a 3D HSE should contain three distinct layers including epidermis, dermis and subcutaneous adipose tissue In addition, cells grown in 3D skin models should form prevalent gap junctions and tight junctions. These subtle cellular structures can enhance the communication of different skin cells, maintain skin tissue integrity and function, and facilitate in vitro drug testing Especially in terms of drug diffusion, drugs in 3D culture models need to diffuse across multiple layers of cells to reach the final targets.
However, this barrier function cannot be found under the 2D culture condition as these subtle structures cannot be maintained on rigid culture dish Nevertheless, most of the traditional 3D skin models still have some serious limitations such as weak barrier properties, lack of vasculature and skin appendages e. Moreover, these 3D skin models cannot offer precise control over spatiotemporal chemical gradients and physical environmental factors e.
Therefore, there is an urgent need to fabricate more physiologically mimicking and functional skin models for drug testing. It can narrow the gap between traditional 2D culture and the in vivo situation, and thus provides the possibility of addressing all these limitations mentioned above. These models have the potential to create functional skin tissues with controlled 3D organization of skin layers and appendages. Herein, in this paper, we begin by providing an overview of some key technologies that are used to construct skin-on-a-chip models.
We then discuss the recent progress on the applications of this type of emerging in vitro skin models for drug testing. Finally, the current challenges and future directions in the development of skin-on-a-chip will be highlighted. For instance, Lee et al. Compared with traditional methods to construct skin tissues, 3D bioprinting offered many advantages such as flexibility, reproducibility, high resolution and high-throughput culture. These engineered skin models can be potentially applied in transdermal and topical formulation discovery, and dermal toxicity studies. In another study, the nanoporous alumina mask was used to fabricate a nanogold platform substrate with its surface nanopatterned with the RGD Arg-Gly-Asp peptide, and two-photon stereolithography techniques were used to manufacture a three-layer-structured cell chip This cell array system can mimic 3D skin cell growth by seeding skin fibroblasts in such designed structures.
Moreover, this device can also achieve high-throughput testing of in vitro effects of cosmetic drugs. More detailed information about such microfabrication techniques applied to fabricating tissue models was reviewed by Verhulsel et al. Taken together, the microfabrication techniques offer the ability to precisely control cell shape, position and 3D organization of skin layers and appendages in a skin-specific context displaying more realistic functionality. However, these microfabricated skin models are still deficient in their ability to recapitulate the human skin due to the lack of several essential cellular or structural components For example, the lack of vascular network in most in vitro skin models cannot simulate in vivo blood circulation in their native counterparts, which is responsible for supplying the living cells with nutrients and growth factors Microfluidics is another core technology to tackle this problem, altering the way we study living skin cells in both the 2D and 3D systems and allowing us to develop a more ideal skin-on-a-chip models When different fluids flow beside each other in the same hollow microchannel, they are entirely laminar and virtually do not mix between neighboring fluids This interesting property enables a small sample volume to be analysed, and thus addresses the limited access to the patient-derived samples and reduces the consumption of the chemicals.
The decrease in the sample size also reduces the amounts of drugs to be tested and results in a higher sensitivity for detecting biomarkers of skin tissues For instance, Mah et al. Due to its miniaturization, this device can be useful in conducting extensive pre-formulation studies for expensive new drugs with limited availability.
Microfluidics-based platforms also permit precise regulation of the cellular microenvironment in skin-on-a-chip models, such as controlling the dynamic fluid behaviours and external physical factors e. Additional examples of such a microfluidic technology applied to skin-on-a-chip models are outlined in subsequent sections. The conventional microfluidic technology combined with the tissue engineering technology has made it possible to engineer more complex skin microsystems.