Conrad, K. M. | bioRxiv Science

bioRxiv2023

Adaptable Fabrication of Vascularized Milliscale Tissues in Membrane-Free Organ Chips Manufactured with 3D Printed Molds

Inexpensive stereolithography (SLA) 3D printing enables rapid prototyping of resin molds for polydimethylsiloxane (PDMS) soft lithography and organ chip fabrication, but geometric distortion and surface roughness of SLA resins can impede the development of adaptable manufacturing workflows. This study reports post-processing procedures for manufacturing SLA-printed molds built with a Formlabs F3 printer that produce fully cured, flat, patently bonded, and optically clear PDMS organ chips. User injection loading tests with iterated guide structure designs were conducted to achieve engineering reduction to practice of milliscale membrane-free organ chips (MFOC), defined as reproducible loading of aqueous solutions without failure of surface tension-based liquid patterning. The optimized manufacturing workflow was applied to further engineer milliscale MFOC for specific applications in modeling vascular physiology and pathobiology. The open lateral interfaces of bulk tissues seeded in MFOC facilitate the formation of anastomoses with internal vasculature to create milliscale perfusable vascular beds. After optimizing bulk tissue vasculogenesis in MFOC, we developed a method for seeding the bulk tissue interfaces with a confluent endothelium to stimulate self-assembly of perfusable anastomoses with the internal vasculature. Rocker- and pump-based flow-conditioning protocols were tested to engineer enhanced barrier function of the perfusable internal vasculature. Modularity of the MFOC design enabled creation of a multi-organ device that was used to model decaying gradients of cancer-associated vascular inflammation in organ compartments positioned at increasing distances from a tumor compartment. These easily adaptable methods for designing and fabricating vascularized microphysiological systems can accelerate their adoption in a diverse range of preclinical laboratory settings.

bioengineering↗

bioRxiv2023

Sex-specific actions of estradiol and testosterone on human fibroblast and endothelial cell proliferation, bioenergetics, and vasculogenesis

Progress toward the development of sex-specific tissue engineered systems has been hampered by the lack of research efforts to define the effects of sex-specific hormone concentrations on relevant human cell types. Here, we investigated the effects of defined concentrations of estradiol (E2) and dihydrotestosterone (DHT) on primary human dermal and lung fibroblasts (HDF and HLF), and human umbilical vein endothelial cells (HUVEC) from female (XX) and male (XY) donors in both 2D expansion cultures and 3D stromal vascular tissues. Sex-matched E2 and DHT stimulation in 2D expansion cultures significantly increased the proliferation index, mitochondrial membrane potential, and the expression of genes associated with bioenergetics (Na+/K+ ATPase, somatic cytochrome C) and beneficial stress responses (chaperonin) in all cell types tested. Notably, cross sex hormone stimulation, i.e., DHT treatment of XX cells in the absence of E2 and E2 stimulation of XY cells in the absence of DHT, decreased bioenergetic capacity and inhibited cell proliferation. We used a microengineered 3D vasculogenesis assay to assess hormone effects on tissue scale morphogenesis. E2 increased metrics of vascular network complexity compared to vehicle in XX tissues. Conversely, and in line with results from 2D expansion cultures, E2 potently inhibited vasculogenesis compared to vehicle in XY tissues. DHT did not significantly alter vasculogenesis in XX or XY tissues but increased the number of non-participating endothelial cells in both sexes. This study establishes a scientific rationale and adaptable methods for using sex hormone stimulation to develop sex-specific culture systems.

bioengineering↗