Bioprinting technology to build a new equivalent skin model with sebaceous gland-like structures
483
Presented by: Caroline Ringenbach
Introduction:
Transition from in-vitro to clinical trials presents a huge gap that has mainly been filled with animal testing. However, testing on animals are not always reliable because they remain physiologically different from human and are banned by the cosmetic industry notably in Europe. Therefore, the need of reliable in vitro models is increasing to test molecules. 3D models for different organs have been set up. These models are physiologically more coherent than 2D models and present advantages in data reliability and are more predictable for clinical trials.
Skin models have been generated first as a graft alternative for patients with severe wounds and burns. These full-skin models have been developed to reproduce a skin equivalent containing human primary fibroblasts embedded in a collagen matrix and human primary keratinocytes that stratify into an epidermis. These models are used in vitro in both medical and cosmetic research. However, they lack skin appendages such as sebaceous glands.
Sebaceous glands are holocrine glands that secrete sebum on the skin surface. Sebum plays a role in hydration, thermoregulation, and microbial protection. 3D models with sebaceous glands consist of skin explants, sebaceous gland explants or 3D skin organoids. Skin explants and isolated sebaceous glands are difficult to procure, hard to cultivate, provide only a limited number of samples, and present donor-donor heterogeneity. There is therefore a need to generate bio-engineered 3D skin models containing sebaceous gland-like structures.
Method used:
Bioprinting has become an essential tool in skin bio-engineering. Bioprinting technologies can be grouped in nozzle-based technologies: micro-valve technology, bio-extrusion and Reactive Jet Impingement (REJI) and nozzle-free technologies: laser-assisted bioprinting (LAB) and sterolithography-based bioprinters. All technologies have advantages and drawbacks such as cost, reliability, speed, resolution, cell viability and handling of printed skin samples due to their size. Hybrid bioprinters combining nozzle-based and laser-assisted bioprinting have major advantages of the afore-mentioned methods and limited drawbacks. We therefore decided to use such multimodal bioprinters to develop a 3D skin model containing structures similar to sebaceous glands.
We first set up sebocyte bioprinting parameters using an affordable and easy to cultivate rodent sebaceous cell line, before switching to a human sebocytes. Since primary sebocytes present a big challenge in terms of cell culture, a new model of sebocytes derived from hiPSCs (human induced Pluripotent Stem Cells) was used. These cells are similar to primary sebocytes in both gene expression modulation and sebum production. They are currently used as a screening tool for sebum production inhibition and induction.
Results
First, we printed spots containing several hundreds of rodent sebocytes on a collagen gel. In each spot, sebocytes generated spheroids of 50-250µm of diameter. To test compatibility with media from skin models, spheroids were cultivated with reconditioned media from different phases of printed skins. We also showed that linoleic acid induced a reorganization of spheroids. Altogether these results validated the feasibility of our system.
We therefore decided to pursue with printing of hiPSC-derived sebocytes. After changing critical bioprinting parameters: (1) number of sebocytes per spot; (2) spot spacing and (3) projection energy of sebocytes, we succeeded printing viable aggregates close to expected size (150µm of diameter).
To assess functionality of our spheroids inside the dermis, we treated them with a compound known for inducing sebogenesis. We observed that FASN (Fatty Acid Synthase), an enzyme that catalyzes de novo fatty acid synthesis, was increased. Models treated with the compound also had more abundant lipid droplets. Furthermore, qPCR quantification of PLIN2 (Perilipin-2), a protein that surrounds lipid droplet envelope, is increased 2X. This validated the functionality of our system.
Finally we have been able to print a full skin model including sebaceous structures.Using confocal and light sheet microscopy, we could characterize these structures as being true spheroids.
Conclusion
This is one very few skin model with sebocytes. It is characterized by bio-printing sebocytes in the dermis of existing 3D skin model, without modulating dermal matrix components.
Despite the quality of these results, we are facing challenges in terms of evaluation of sebum production. Indeed, evaluation of effectors on sebum production by IMF are dependent of cutting area. Light-sheet microscopy is a nice but expensive alternative.
Further studies will be conducted to go deeper into the characterization of these promising models
Transition from in-vitro to clinical trials presents a huge gap that has mainly been filled with animal testing. However, testing on animals are not always reliable because they remain physiologically different from human and are banned by the cosmetic industry notably in Europe. Therefore, the need of reliable in vitro models is increasing to test molecules. 3D models for different organs have been set up. These models are physiologically more coherent than 2D models and present advantages in data reliability and are more predictable for clinical trials.
Skin models have been generated first as a graft alternative for patients with severe wounds and burns. These full-skin models have been developed to reproduce a skin equivalent containing human primary fibroblasts embedded in a collagen matrix and human primary keratinocytes that stratify into an epidermis. These models are used in vitro in both medical and cosmetic research. However, they lack skin appendages such as sebaceous glands.
Sebaceous glands are holocrine glands that secrete sebum on the skin surface. Sebum plays a role in hydration, thermoregulation, and microbial protection. 3D models with sebaceous glands consist of skin explants, sebaceous gland explants or 3D skin organoids. Skin explants and isolated sebaceous glands are difficult to procure, hard to cultivate, provide only a limited number of samples, and present donor-donor heterogeneity. There is therefore a need to generate bio-engineered 3D skin models containing sebaceous gland-like structures.
Method used:
Bioprinting has become an essential tool in skin bio-engineering. Bioprinting technologies can be grouped in nozzle-based technologies: micro-valve technology, bio-extrusion and Reactive Jet Impingement (REJI) and nozzle-free technologies: laser-assisted bioprinting (LAB) and sterolithography-based bioprinters. All technologies have advantages and drawbacks such as cost, reliability, speed, resolution, cell viability and handling of printed skin samples due to their size. Hybrid bioprinters combining nozzle-based and laser-assisted bioprinting have major advantages of the afore-mentioned methods and limited drawbacks. We therefore decided to use such multimodal bioprinters to develop a 3D skin model containing structures similar to sebaceous glands.
We first set up sebocyte bioprinting parameters using an affordable and easy to cultivate rodent sebaceous cell line, before switching to a human sebocytes. Since primary sebocytes present a big challenge in terms of cell culture, a new model of sebocytes derived from hiPSCs (human induced Pluripotent Stem Cells) was used. These cells are similar to primary sebocytes in both gene expression modulation and sebum production. They are currently used as a screening tool for sebum production inhibition and induction.
Results
First, we printed spots containing several hundreds of rodent sebocytes on a collagen gel. In each spot, sebocytes generated spheroids of 50-250µm of diameter. To test compatibility with media from skin models, spheroids were cultivated with reconditioned media from different phases of printed skins. We also showed that linoleic acid induced a reorganization of spheroids. Altogether these results validated the feasibility of our system.
We therefore decided to pursue with printing of hiPSC-derived sebocytes. After changing critical bioprinting parameters: (1) number of sebocytes per spot; (2) spot spacing and (3) projection energy of sebocytes, we succeeded printing viable aggregates close to expected size (150µm of diameter).
To assess functionality of our spheroids inside the dermis, we treated them with a compound known for inducing sebogenesis. We observed that FASN (Fatty Acid Synthase), an enzyme that catalyzes de novo fatty acid synthesis, was increased. Models treated with the compound also had more abundant lipid droplets. Furthermore, qPCR quantification of PLIN2 (Perilipin-2), a protein that surrounds lipid droplet envelope, is increased 2X. This validated the functionality of our system.
Finally we have been able to print a full skin model including sebaceous structures.Using confocal and light sheet microscopy, we could characterize these structures as being true spheroids.
Conclusion
This is one very few skin model with sebocytes. It is characterized by bio-printing sebocytes in the dermis of existing 3D skin model, without modulating dermal matrix components.
Despite the quality of these results, we are facing challenges in terms of evaluation of sebum production. Indeed, evaluation of effectors on sebum production by IMF are dependent of cutting area. Light-sheet microscopy is a nice but expensive alternative.
Further studies will be conducted to go deeper into the characterization of these promising models