Skin is the largest organ of the body and is susceptible to various ailments due to constant interactions with the outer harsh environment. Skin diseases can arise from genetic disorders as well as a manifestation of underlying disease conditions. We aim to develop stable vascularized, immunocompetent full-thickness skin models. Our approach angles out from the conventional two-dimensional monolayer models and hydrogel based 3D skin models. To achieve this, we focus on the development of scaffold-free skin models via ECM coating and accumulation technique where single cells are coated layer-by-layer with ECM moieties and subsequently cultivated to acquire thick scaffold-free tissue.
Figure 1. The transition from 2-D to 3-D to dynamic 3-D culture: A) Monolayer Keratinocytes cultured on cell culture inserts (red=actin), B) Scaffold-free 3-D full thickness vascularized skin model shows optimal epidermal differentiation (H&E staining), C) Transition to a custom 3-D printed bioreactor that provides both submerged and air-liquid culture, D) Formation of stable vasculature within the skin model (red=CD31 stain), E) Dynamic flow leads to vessel morphogenesis and migration towards the flow source (red=CD31, grey=DAPI), F) Fluorescent beads (blue) observed within the blood vessels (red) in flow culture (green=actin). Image revised from Applied Materials Today, 2021
Smriti Singh
Figure 1. The transition from 2-D to 3-D to dynamic 3-D culture: A) Monolayer Keratinocytes cultured on cell culture inserts (red=actin), B) Scaffold-free 3-D full thickness vascularized skin model shows optimal epidermal differentiation (H&E staining), C) Transition to a custom 3-D printed bioreactor that provides both submerged and air-liquid culture, D) Formation of stable vasculature within the skin model (red=CD31 stain), E) Dynamic flow leads to vessel morphogenesis and migration towards the flow source (red=CD31, grey=DAPI), F) Fluorescent beads (blue) observed within the blood vessels (red) in flow culture (green=actin). Image revised from Applied Materials Today, 2021
Smriti Singh
Dynamic flow culture
The vasculature is one of the biggest challenges in tissue engineering. While much work in this regard has been done to induce angiogenesis, our efforts are also directed towards a lesser-known, yet important issue i.e. to sustain the blood vessels for the long term. Blood vessels undergo abnormal angiogenesis or regression in lab-grown tissue due to the abundance or scarcity of growth factors, ECM, and cellular types. To sustain the vascular integrity within the tissue models, we utilize custom 3-D printed bioreactors that provide optimal biomechanical cues to sustain vasculature. We aim to further explore different bioreactor designs affecting flow patterns and shear to obtain more perfused vasculature and overall physiological tissue architecture.
Figure 2 A) Bioreactor design B) flow streamline visualization in the entire bioreactor C)flow streamline visualization through the cell culture insert and D) through the bioreactor well respectively.
Smriti Singh
Figure 2 A) Bioreactor design B) flow streamline visualization in the entire bioreactor C)flow streamline visualization through the cell culture insert and D) through the bioreactor well respectively.
Smriti Singh
Modeling of skin disease
Figure 3. In-vitro Psoriasis models. Similar to in-vivo clinical scenario, we provide the skin models with IL-17, mimicking the release of IL-17 from T-cells. The addition of IL-17 leads to the formation of psoriasis-like phenotype. Sekukinumab, an anti-IL17 antibody was tested on psoriatic skin models in order to observe the remission and study the changes in barrier property-related and inflammatory-related genes.
Smriti Singh
Figure 3. In-vitro Psoriasis models. Similar to in-vivo clinical scenario, we provide the skin models with IL-17, mimicking the release of IL-17 from T-cells. The addition of IL-17 leads to the formation of psoriasis-like phenotype. Sekukinumab, an anti-IL17 antibody was tested on psoriatic skin models in order to observe the remission and study the changes in barrier property-related and inflammatory-related genes.
Smriti Singh
With the evolving field of skin tissue engineering, we aim to fabricate skin models to study different skin diseases. We showed that scaffold-free skin models can be cultivated for long duration (49 days). Owing to the longevity and robustness of the skin model, we could mimic Psoriasis, a chronic skin disease by addition of IL-17 followed by the testing of anti-IL17 antibody Sekukinumab in presence of IL-17 on the psoriatic models. We also currently mimic and study other skin diseases such as Atopic dermatitis (eczema) and Pemphigus to expand the application of scaffold-free skin models.
Alveolar Capillary Model
We work on establishing an alveolar-capillary barrier model of human primary cells supported by fully synthetic bio-functional nanofibers basement membrane (BM) mimics. The mimic resembled the fibrous and ultra-thin nature (2µm) of the natural BM. This facilitated bipolar cultivation of endothelial and epithelial cells with fundamental alveolar functionality and allowed the chemotaxis of neutrophils across the barrier.
Figure 4. Schematic illustration of the fabrication process of the alveolar barrier model. (a) Illustrated in the scheme are the chemical structures of polymers used in this study. NCO-sPEG with hydrophilic chains and NCO end groups for conjugation of biomolecules and bioresorbable PCL. (b) Nanofiber mesh as a BM mimic was formed by electrospun nanofibers of PCL-sPEG functionalized with biomolecules. (c) Human primary epithelial and endothelial cells were bipolar-cultured on nanofiber mesh to reconstruct an alveolar barrier model capable of cell−cell crosstalk (Adapted from Nishiguchi et al, Biomacromolecules 2018.).
Smriti Singh
Figure 4. Schematic illustration of the fabrication process of the alveolar barrier model. (a) Illustrated in the scheme are the chemical structures of polymers used in this study. NCO-sPEG with hydrophilic chains and NCO end groups for conjugation of biomolecules and bioresorbable PCL. (b) Nanofiber mesh as a BM mimic was formed by electrospun nanofibers of PCL-sPEG functionalized with biomolecules. (c) Human primary epithelial and endothelial cells were bipolar-cultured on nanofiber mesh to reconstruct an alveolar barrier model capable of cell−cell crosstalk (Adapted from Nishiguchi et al, Biomacromolecules 2018.).
Smriti Singh
3-D Tumor models
To study the indirect interaction of bone-cells (secondary site) and vascularized breast cancer microenvironment (primary site) in the progression of metastasis, we fabricated a three-dimensional vascularized breast cancer model. The scaffold-free vascularized breast cancer tissues were composed of MDA-MB231 cells distributed in the fibroblastic tissue and in proximity with the blood vessel. Inclusion of osteoblasts in the presence of breast cancer cells led to changes in cancer migration, blood vessel architecture and levels of growth factors. Comparing the models cultured with and without the osteoblasts, we show several interesting upregulated hub genes from the gene array data linking to poor prognosis in breast cancer patients. Our study strongly indicates the role of osteoblast in breast cancer metastasis progression and highlights the suitability of the 3-D in vitro model to study pathways involved in metastasis and organotropism. We aim to fabricate different tumor models (breast, pancreatic, skin, lung) to engineer a more realistic tumor environment by utilizing scaffold-free and different scaffold-based techniques.
Figure 5 shows the distribution of triple negative breast cancer (MDA-MB231, green) in a scaffold-free vascularized tissue. Vessels are stained with CD31 (red). Breast cancer cells form a highly elongated morphology in scaffold-free tissues as the fibroblast-derived natural-ECM provides an optimal microenvironment for the cancer cells.
Smriti Singh
Figure 5 shows the distribution of triple negative breast cancer (MDA-MB231, green) in a scaffold-free vascularized tissue. Vessels are stained with CD31 (red). Breast cancer cells form a highly elongated morphology in scaffold-free tissues as the fibroblast-derived natural-ECM provides an optimal microenvironment for the cancer cells.
Smriti Singh
1. S. Singh*, Y. Marquardt, R. Rimal, A. Nishiguchi, S. Huth, M. Akashi, M. Moeller, J. M. Baron*, ACS Applied Bio Materials 2020, 3, 6639.
2. R. Rimal, Y. Marquardt, T. Nevolianis, S. Djeljadini, A. B. Marquez, S. Huth, D. N. Chigrin, M. Wessling, J. M. Baron, M. Möller, S.Singh* Applied Materials Today 2021, 25, 101213.
3.Jain, P.; Nishiguchi, A.; Linz, G.; Wessling, M.; Ludwig, A.; Rossaint, R.; Möller, M.; Singh, S.*, Reconstruction of Ultra-thin Alveolar-capillary Basement Membrane Mimics. Advanced biology 2021, 5 (8), e2000427
4. Rimal, R.; Desai, P.; Marquez, A. B.; Sieg, K.; Marquardt, Y.; Singh, S.*, 3-D vascularized breast cancer model to study the role of osteoblast in formation of a pre-metastatic niche. Scientific Reports 2021, 11 (1).
