Cultivation of hierarchical 3D scaffolds inside a perfusion bioreactor: scaffold design and finite-element analysis of fluid flow

The use of porous 3D scaffolds for the repair of bone nonunion and osteoporotic bone is currently an area of great interest. Using a combination of thermally-induced phase separation (TIPS) and 3D-plotting (3DP), we have generated hierarchical 3DP/TIPS scaffolds made of poly(lactic-co-glycolic acid) (PLGA) and nanohydroxyapatite (nHA). A full factorial design of experiments was conducted, in which the PLGA and nHA compositions were varied between 6‒12% w/v and 10‒40% w/w, respectively, totaling 16 scaffold formulations with an overall porosity ranging between 87%‒93%. These formulations included an optimal scaffold design identified in our previous study. The internal structures of the scaffolds were examined using scanning electron microscopy and microcomputed tomography. Our optimal scaffold was seeded with MC3T3-E1 murine preosteoblastic cells and subjected to cell culture inside a tissue culture dish and a perfusion bioreactor. The results were compared to those of a commercial CellCeram™ scaffold with a composition of 40% β-tricalcium phosphate and 60% hydroxyapatite (β-TCP/HA). Media flow within the macrochannels of 3DP/TIPS scaffolds was modeled in COMSOL software in order to fine tune the wall shear stress. CyQUANT DNA assay was performed to assess cell proliferation. The normalized number of cells for the optimal scaffold was more than twofold that of CellCeram™ scaffold after two weeks of culture inside the bioreactor. Despite the substantial variability in the results, the observed improvement in cell proliferation upon culture inside the perfusion bioreactor (vs. static culture) demonstrated the role of macrochannels in making the 3DP/TIPS scaffolds a promising candidate for scaffold-based tissue engineering.

Abstract P383: 3D-collagen Matrix Enhanced Integrins And Matrix Metalloproteinases Expression In Cardiac Mesenchymal Cells

Introduction: Use of cardiac mesenchymal cells (CMCs) has been shown to improve cardiac function following myocardial infarction. Main drawback in cardiac cell therapy is the major loss of injected cells within few hours. Increase the retention of these injected cells could increase their efficacy, where cardiac patches with various cell types showed better outcome. Among, collagen patch plays lead role as a cell-laden matrix in cardiac tissue engineering. Creating a detailed understanding of how collagen matrix changes the cellular phenotype could provide seminal insights to regeneration therapy. Hypothesis: Growing CMCs in three dimensional (3D) collagen matrix could alter the expression of extracellular matrix (ECM) and adhesion molecules, which may enhance their efficacy. Methods: The bovine type I collagen was chemically modified and solubilized in culture medium with photo-initiator. The mouse CMCs were isolated and resuspended in collagen solution, printed using 3D bioprinter and UV-crosslinked to form 3D-CMC construct. The 3D-CMC construct was submerged in growth medium and cultured for 48h and analyzed for the expression of ECM and adhesion molecules (n=5/group). CMCs cultured in regular plastic tissue culture dish was used as control. Results: RT profiler array showed changes in the ECM and adhesion molecules expression, specifically certain integrins and matrix metalloproteinases (MMPs) in CMCs cultured 3D collagen construct compared to 2D monolayer. Subsequent qRT-PCR analysis revealed significant (p<0.01) upregulation of integrins such as Itga2 (2.96±0.13), Itgb1 (3.18±0.2) and Itgb3 (2.4±0.27) and MMPs such as MMP13 (37.2±3.36), MMP9 (5.23±1.06) and MMP3 (7.14±2.07). Western blot analysis further confirmed significant elevation of these integrins and matrix metalloproteinases at protein level. Collagen encapsulation did not alter the expression of N-cadherin in CMCs, which is a potential mesenchymal cadherin adhesion molecule. Conclusion: Integrin αβ heterodimers transduce signals that facilitate cell homing, migration, survival and differentiation. Similarly, MMPs plays vital role in cell migration and proliferation. Our results demonstrate that the 3D-collagen Niche enhances the expression of certain integrins and MMPs in CMCs. This suggest that the efficacy of CMCs could be magnified by providing 3D architecture with collagen matrix and further in vivo experiments would reveal functional benefits from CMCs for clinical use.

An Improved Transwell Design for Microelectrode Ion-Flux Measurements

The microelectrode ion flux estimation (MIFE) is a powerful, non-invasive electrophysiological method for cellular membrane transport studies. Usually, the MIFE measurements are performed in a tissue culture dish or directly with tissues (roots, parts of the plants, and cell tissues). Here, we present a transwell system that allows for MIFE measurements on a cell monolayer. We introduce a measurement window in the transwell insert membrane, which provides direct access for the cells to the media in the upper and lower compartment of the transwell system and allows direct cell-to-cell contact coculture. Three-dimensional multiphoton lithography (MPL) was used to construct a 3D grid structure for cell support in the measurement window. The optimal polymer grid constant was found for implementation in transwell MIFE measurements. We showed that human umbilical vein endothelial cells (HUVECs) efficiently grow and maintain their physiological response on top of the polymer structures.

Viral Plaque Assay v1

This is part 3.6 of the "Study of MAIT Cell Activation in Viral Infections In Vivo" collection of protocols. Collection Abstract: MAIT cells are abundant, highly evolutionarily conserved innate-like lymphocytes expressing a semi-invariant T cell receptor (TCR), which recognizes microbially derived small intermediate molecules from the riboflavin biosynthetic pathway. However, in addition to their TCR-mediated functions they can also be activated in a TCR-independent manner via cytokines including IL-12, -15, -18, and type I interferon. Emerging data suggest that they are expanded and activated by a range of viral infections, and significantly that they can contribute to a protective anti-viral response. Here we describe methods used to investigate these anti-viral functions in vivo in murine models. To overcome the technical challenge that MAIT cells are rare in specific pathogen-free laboratory mice, we describe how pulmonary MAIT cells can be expanded using intranasal bacterial infection or a combination of synthetic MAIT cell antigen and TLR agonists. We also describe protocols for adoptive transfer of MAIT cells, methods for lung homogenization for plaque assays, and surface and intracellular cytokine staining to determine MAIT cell activation. Abstract: Viral plaque assays are used to determine influenza viral titers. A diluted solution of egg-adapted Influenza A viruses/lung-infected tissue homogenates are applied to a six-well tissue culture dish containing a monolayer of Madin-Darby canine kidney (MDCK) cells. The infected MDCK cells grow under a semisolid overlay medium (agar) containing trypsin. A plaque is produced when a virus particle infects a cell, replicates, and then kills the cell. This process can be repeated several times as surrounding cells can be infected by newly replicated virus and killed. When visualized by eye, plaques appear as white spots. The assay is measured in PFU/mL.

hPSC-derived organoids: models of human development and disease

Organoids derived from human pluripotent stem cells (hPSCs) have emerged as important models for investigating human-specific aspects of development and disease. Here we discuss hPSC-derived organoids through the lens of development—highlighting how stages of human development align with the development of hPSC-derived organoids in the tissue culture dish. Using hPSC-derived lung and intestinal organoids as examples, we discuss the value and application of such systems for understanding human biology, as well as strategies for enhancing organoid complexity and maturity.

Natural Formulations Provide Antioxidant Complement to Hyaluronic Acid-Based Topical Applications Used in Wound Healing

Hyaluronic acid (HA) promotes wound healing, and, accordingly, formulations based on HA have been widely used in regenerative medicine. In addition, naturally derived compounds, e.g., plant-based extracts and vitamin E, have exhibited antioxidant activity. In this study, a formulation containing hyaluronic acid, vitamin E, raspberry extract, and green tea was developed for potential topical applications, targeting wound healing. Rheological analysis was performed along with antioxidant and biological studies. The rheological characterization showed that the HA-based formulation is a thixotropic platform and possesses higher mechanical properties than the control formulation. To evaluate the wound healing potential of the formulation, an in vitro “wound healing” assay was carried out using human derived fibroblasts (HDF) with a cell-free gap on the tissue culture dish. The formulation showed better wound healing ability than the control formulation.

Mesenchymal Cell Growth and Differentiation on a New Biocomposite Material: A Promising Model for Regeneration Therapy

Mesenchymal stem cells serve as the body’s reservoir for healing and tissue regeneration. In cases of severe tissue trauma where there is also a need for tissue organization, a scaffold may be of use to support the cells in the damaged tissue. Such a scaffold should be composed of a material that can biomimic the mechanical and biological properties of the target tissues in order to support autologous cell-adhesion, their proliferation, and differentiation. In this study, we developed and assayed a new biocomposite made of unique collagen fibers and alginate hydrogel that was assessed for the ability to support mesenchymal cell-proliferation and differentiation. Analysis over 11 weeks in vitro demonstrated that the scaffold was biocompatible and supports the cells viability and differentiation to produce tissue-like structures or become adipocyte under differentiation medium. When the biocomposite was enriched with nano particles (NPs), mesenchymal cells grew well after uptake of fluorescein isothiocyanate (FITC) labeled NPs, maintained their viability, migrated through the biocomposite, reached, and adhered to the tissue culture dish. These promising findings revealed that the scaffold supports the growth and differentiation of mesenchymal cells that demonstrate their full physiological function with no sign of material toxicity. The cells’ functionality performance indicates and suggests that the scaffold is suitable to be developed as a new medical device that has the potential to support regeneration and the production of functional tissue.

67 Effects of phytohemagglutinin on the culture of isolated bovine blastomeres derived from the 8-cell stage invitro-produced embryos

Monozygotic twin embryos which can efficiently be produced by blastomere separation and aggregation of early cleavage stages of embryos using commercially provided well-of-the-well (WOW) culture dish. Phytohaemagglutinin (PHA) is a plant lectin that binds to and aggregates on the surface of animal cells, but also contains toxicity that causes food poisoning. The present study was conducted to evaluate the toxicity to embryos and the effect to development of isolated blastomeres on PHA-supplemented WOW culture. Embryos were produced using oocytes from ovaries collected at an abattoir by IVM, IVF, and invitro culture (IVC). The tissue culture medium 199 supplemented with 5% calf serum (CS), Brackett-Oliphant solution supplemented with 10mgmL−1 bovine serum albumin, and CR1aa medium containing 5% CS were used for each culture step. For the evaluation of PHA toxicity, 89 embryos that developed to the 5-8-cell stage were obtained at Day 2 after insemination. Each embryo was cultured in a droplet of 5 µL/embryo IVC culture medium supplemented with or without PHA. For the evaluation of PHA to development of isolated blastomeres, 111 of 8-cell stage embryos were obtained 48-54h post-insemination. Zonae pellucidae were removed by exposure to 0.25% pronase. Then, embryos were separated into single blastomeres by gentle pipetting in IVC medium. Each four blastomeres were formed in the shape of a bunch inside the thin cylinder at the tip of the Pasteur pipette by gentle pipetting. Then, each mass of blastomeres in each 60 masses was cultured individually in 5-µL droplets of IVC medium supplemented with or without PHA on the flat surface of a tissue culture dish. On the other hand, each four blastomeres were introduced into a single conical micro-well each having a diameter and depth of ~287µm and 168µm (Dai Nippon Printing). This culture of blastomeres was performed covered with a droplet of 2.5µL well−1 IVC medium supplemented with or without PHA in each 50 or 52 wells. In all of investigations, PHA was used at 50µgmL−1 (Akagi et al. 2011 J. Reprod. Dev. 57). Statistical analysis was performed using Student's t-test and analysis of variance. The blastocyst formation rate (71.1±2.3% vs. 72.7±1.7%), total cell number (120 vs. 122), and inner cell mass cell number (47 vs. 51) at Day 7 after IVF did not differ between PHA-supplemented and PHA-free group in the toxicity test, respectively. In the blastomere culture, the blastocyst formation rate was very low (10.0±5.9% vs. 5.0±2.9%) regardless of the PHA supplementation in drops on the flat surface of a tissue culture dish. On the other hand, blastocyst formation was improved using the WOW culture dish (24.0±3.6% vs. 40.4±7.6%) but there was no difference with or without PHA supplementation. Although nontoxicity of PHA and efficacy of WOW culture for isolated-aggregated blastomeres were confirmed, no improvement of PHA supplementation on development was observed in this study. Subsequently, experiments on the optimum concentration of PHA for aggregation and development of blastomeres in WOW culture are required.

The para-fluoro-thiol ligation in water

The para-fluoro-thiol ligation is performed for the first time in aqueous medium and shown to be controlled by pH. Solution kinetics in various conditions of pH, temperature, and concentration are reported, together with an application for the modification of a polymeric tissue culture dish with a peptide.