Engineered 3D Hydrogel Culture Environments to Investigate Trophoblast Differentiation and Immunomodulation

193630-Thumbnail Image.png
Description
The human placenta is comprised of trophoblast subtypes – cytotrophoblasts, syncytiotrophoblasts (ST), and extravillous trophoblasts (EVT) – that are crucial for successful pregnancies. Understanding trophoblast functions is essential to treat pregnancy complications and investigate maternal-fetal immune tolerance. Trophoblasts secrete factors

The human placenta is comprised of trophoblast subtypes – cytotrophoblasts, syncytiotrophoblasts (ST), and extravillous trophoblasts (EVT) – that are crucial for successful pregnancies. Understanding trophoblast functions is essential to treat pregnancy complications and investigate maternal-fetal immune tolerance. Trophoblasts secrete factors to instruct tolerance; however, the distinct trophoblast subtypes’ role in fetal tissue tolerance remain insufficiently understood. Inadequate models to study the human placenta in vitro limit the current understanding of human placental behavior and development. Synthetic hydrogel systems such as poly(ethylene) glycol (PEG)-maleimide offer a highly defined, tunable 3D environment to study trophoblast subtypes, which may overcome experimental variability in naturally derived hydrogels like Matrigel due to batch-to-batch variability. Here, a PEG hydrogel system with tunable degradability and placenta-derived extracellular matrix ligands is employed to evaluate the capacity of the hydrogel library to support the function and phenotypic protein expression of three trophoblast-like cell lines, assess the differentiation of trophoblast stem cells (TSC), and explore the effects of trophoblast supernatants on the modulation of protein expression and secretion by immune cell subsets present at the maternal-fetal interface. Degradable synthetic hydrogels support the greatest degree of trophoblast-like spheroid proliferation and function relative to standard Matrigel controls and culture conditions modulate trophoblast-like and TSC functional subtype as measured by proteomics analysis and functional secretion assays. PEG hydrogels support TSC viability and function comparable to Matrigel; however, ST-differentiated cells prefer PEG environments, while EVT-differentiated cells favor Matrigel, as assessed through phenotypic protein expression and secretion. These data highlight the influence of trophoblast culture condition and subtype on immune cell protein expression and inflammatory cytokine secretion in response to trophoblast supernatants. This model advances the understanding of in vitro placental modeling, which can provide insights on pregnancy-related complications and maternal-fetal immunotolerance.
Date Created
2024
Agent

Development of a Hydrogel Macroencapsulation Device for Improved Long-Term Islet Survival Using Injection Molding and Oxygen Modeling-Aided Design

190904-Thumbnail Image.png
Description
Allogeneic islet transplantation has the potential to reverse Type 1 Diabetes in patients. However, limitations such as chronic immunosuppression, islet donor numbers, and islet survival post-transplantation prevent the widespread application of allogeneic islet transplantation as the treatment of choice. Macroencapsulation

Allogeneic islet transplantation has the potential to reverse Type 1 Diabetes in patients. However, limitations such as chronic immunosuppression, islet donor numbers, and islet survival post-transplantation prevent the widespread application of allogeneic islet transplantation as the treatment of choice. Macroencapsulation devices have been widely used in allogeneic islet transplantation due to their capability to shield transplanted cells from the immune system as well as provide a supportive environment for cell viability, but macroencapsulation devices face oxygen transport challenges as their geometry increases from preclinical to clinical scales. The goal of this work is to generate complex 3D hydrogel macroencapsulation devices with sufficient oxygen transport to support encapsulated cell survival and generate these devices in a way that is accessible in the clinic as well as scaled manufacturing. A 3D-printed injection mold has been developed to generate hydrogel-based cell encapsulation devices with spiral geometries. The spiral geometry of the macroencapsulation device facilitates greater oxygen transport throughout the whole device resulting in improved islet function in vivo in a syngeneic rat model. A computational model of the oxygen concentration within macroencapsulation devices, validated by in vitro analysis, predicts that cells and islets maintain a greater viability and function in the spiral macroencapsulation device. To further validate the computational model, pO2 Reporter Composite Hydrogels (PORCH) are engineered to enable spatiotemporal measurement of oxygen tension within macroencapsulation devices using the Proton Imaging of Siloxanes to map Tissue Oxygenation Levels (PISTOL) magnetic resonance imaging approach. Overall, a macroencapsulation device geometry designed via computational modeling of device oxygen gradients and validated with magnetic resonance (MR) oximetry imaging enhances islet function and survival for islet transplantation.
Date Created
2023
Agent

Characterization of a Low-Cost, Open-Source Nanoprecipitation Method for Fabrication of Polyester Nanoparticles

Description

Polymeric nanoparticles (NP) consisting of Poly Lactic-co-lactic acid - methyl polyethylene glycol (PLLA-mPEG) or Poly Lactic-co-Glycolic Acid (PLGA) are an emerging field of study for therapeutic and diagnostic applications. NPs have a variety of tunable physical characteristics like size, morphology,

Polymeric nanoparticles (NP) consisting of Poly Lactic-co-lactic acid - methyl polyethylene glycol (PLLA-mPEG) or Poly Lactic-co-Glycolic Acid (PLGA) are an emerging field of study for therapeutic and diagnostic applications. NPs have a variety of tunable physical characteristics like size, morphology, and surface topography. They can be loaded with therapeutic and/or diagnostic agents, either on the surface or within the core. NP size is an important characteristic as it directly impacts clearance and where the particles can travel and bind in the body. To that end, the typical target size for NPs is 30-200 nm for the majority of applications. Fabricating NPs using the typical techniques such as drop emulsion, microfluidics, or traditional nanoprecipitation can be expensive and may not yield the appropriate particle size. Therefore, a need has emerged for low-cost fabrication methods that allow customization of NP physical characteristics with high reproducibility. In this study we manufactured a low-cost (<$210), open-source syringe pump that can be used in nanoprecipitation. A design of experiments was utilized to find the relationship between the independent variables: polymer concentration (mg/mL), agitation rate of aqueous solution (rpm), and injection rate of the polymer solution (mL/min) and the dependent variables: size (nm), zeta potential, and polydispersity index (PDI). The quarter factorial design consisted of 4 experiments, each of which was manufactured in batches of three. Each sample of each batch was measured three times via dynamic light scattering. The particles were made with PLLA-mPEG dissolved in a 50% dichloromethane and 50% acetone solution. The polymer solution was dispensed into the aqueous solution containing 0.3% polyvinyl alcohol (PVA). Data suggests that none of the factors had a statistically significant effect on NP size. However, all interactions and relationships showed that there was a negative correlation between the above defined input parameters and the NP size. The NP sizes ranged from 276.144 ± 14.710 nm at the largest to 185.611 ± 15.634 nm at the smallest. In conclusion, the low-cost syringe pump nanoprecipitation method can achieve small sizes like the ones reported with drop emulsion or microfluidics. While there are trends suggesting predictable tuning of physical characteristics, significant control over the customization has not yet been achieved.

Date Created
2023-05
Agent

Engineering a Tolerogenic Immunomodulatory Hydrogel

168426-Thumbnail Image.png
Description
Placental pregnancy is a biological scenario where tissue types bearing different antigen signatures co-exist within the same microenvironment without rejection. Placental trophoblast cells locally modulate the immune system in pregnancy, and one process through which this occurs is through the

Placental pregnancy is a biological scenario where tissue types bearing different antigen signatures co-exist within the same microenvironment without rejection. Placental trophoblast cells locally modulate the immune system in pregnancy, and one process through which this occurs is through the release of a class of nano-scaled extracellular vesicles called exosomes. The aim is to use these placental-derived immunomodulatory exosomes as a therapeutic and engineer a means to deliver these exosomes using a hydrogel vehicle. As such, two representative trophoblast cell lines, JAR and JEG-3, were used as exosome sources. First step involved the evaluation of the morphological and proteomic characterization of the isolated exosomes through dynamic light scattering (DLS) analysis, transmission electron microscopy (TEM) imaging, and mass spectrometry (MS) analysis. Following exosome characterization, incorporation of exosomes within hydrogel matrices like polyethylene glycol and alginate to determine their release profile over a timescale of 14 days was performed. Comparing the release between the two cell lines isolated exosomes, no discernible difference is observed in their release, and release appears complete within two days. Future studies will evaluate the impact of exosome loadings and hydrogel modification on exosome release profiles, as well as their influence on immune cells.
Date Created
2021
Agent