Pumpless microfluidic platform for drug testing on human skin equivalents

Abstract

Advances in bio-mimetic in vitro human skin models increase the efficiency of drug screening studies. In this study, we designed and developed a microfluidic platform that allows for long-term maintenance of full thickness human skin equivalents (HSE) which are comprised of both the epidermal and dermal compartments. The design is based on the physiologically relevant blood residence times in human skin tissue and allows for the establishment of an air-epidermal interface which is crucial for maturation and terminal differentiation of HSEs. The small scale of the design reduces the amount of culture medium and the number of cells required by 36 fold compared to conventional transwell cultures. Our HSE-on-a-chip platform has the capability to recirculate the medium at desired flow rates without the need for pump or external tube connections. We demonstrate that the platform can be used to maintain HSEs for three weeks with proliferating keratinocytes similar to conventional HSE cultures. Immunohistochemistry analyses show that the differentiation and localization of keratinocytes was successfully achieved, establishing all sub-layers of the epidermis after one week. Basal keratinocytes located at the epidermal-dermal interface remain in a proliferative state for three weeks. We use a transdermal transport model to show that the skin barrier function is maintained for three weeks. We also validate the capability of the HSE-on-a-chip platform to be used for drug testing purposes by examining the toxic effects of doxorubucin on skin cells and structure. Overall, the HSE-on-a-chip is a user-friendly and cost-effective in vitro platform for drug testing of candidate molecules for skin disorders.

Fig. 1. Description of the microfluidic system and transport model

(A) Scheme of the HSE-on-a-chip platform with each individual component (B) Description of the transport model geometry and parameters.

Fig. 2. The duration of maintenance of HSEs-on-a-chip

(A) H&E staining of HSEs maintained HSE-on-a-chip or conventional cultures for 4 weeks, (B) Ki67 immunostaining of HSEs-on-a-chip to evaluate proliferation activity of basal keratinocytes. Scale bars: 100 µm

Fig. 3. Long-term maintenance of HSEs-on-a-chip

(A)Immunolabeled HSE-on-a-chip and control samples with layer specific markers, Keratin-1, Keratin-14 and Loricrin (green) and DAPI (blue) showing proper formation and structural integrity of epidermal layers after 3 weeks. Scale bars: 100 µm (B) Transdermal transport of FAM-tagged oligonucleotides through HSE-on-a-chip in comparison to polycarbonate membrane. The overall permeability coefficients, K_p, of the HSEs were estimated using the transport model to evaluate the skin barrier function throughout 3 weeks.

Fig. 4. The toxicity of doxorubucin (36 µM) on HSE-on-a-chip

(A) H&E staining of the HSE-on-a-chip after one week of doxorubucin treatment in comparison to untreated HSE-on-a-chip. (B) Immunostaining of Ki67 and Loricrin of HSE-on-a-chip treated with doxorubicin for a week. Scale bars: 100 µm