Gelatin-methacryloyl (GelMA) is a semi-synthetic hydrogel which consists of gelatin derivatized with methacrylamide and methacrylate groups. These hydrogels provide cells with an optimal biological environment (e.g., RGD motifs for adhesion) and can be quickly photo-crosslinked, which provides shape fidelity and stability at physiological temperature. In the present work, we demonstrated how GelMA hydrogels can be synthesized with a specific degree of functionalization (DoF) and adjusted to the intended application as a three-dimensional (3D) cell culture platform. The focus of this work lays on producing hydrogel scaffolds which provide a cell promoting microenvironment for human adipose tissue-derived mesenchymal stem cells (hAD-MSCs) and are conductive to their adhesion, spreading, and proliferation. The control of mechanical GelMA properties by variation of concentration, DoF, and ultraviolet (UV) polymerization conditions is described. Moreover, hAD-MSC cell viability and morphology in GelMA of different stiffness was evaluated and compared. Polymerized hydrogels with and without cells could be digested in order to release encapsulated cells without loss of viability. We also demonstrated how hydrogel viscosity can be increased by the use of biocompatible additives, in order to enable the extrusion bioprinting of these materials. Taken together, we demonstrated how GelMA hydrogels can be used as a versatile tool for 3D cell cultivation.
Following cultivation of distinct mesenchymal stem cell (MSC) populations derived from human umbilical cord under hypoxic conditions (between 1.5% to 5% oxygen (O2)) revealed a 2- to 3-fold reduced oxygen consumption rate as compared to the same cultures at normoxic oxygen levels (21% O2). A simultaneous measurement of dissolved oxygen within the culture media from 4 different MSC donors ranged from 15 μmol/L at 1.5% O2to 196 μmol/L at normoxic 21% O2. The proliferative capacity of the different hypoxic MSC populations was elevated as compared to the normoxic culture. This effect was paralleled by a significantly reduced cell damage or cell death under hypoxic conditions as evaluated by the cellular release of LDH whereby the measurement of caspase3/7 activity revealed little if any differences in apoptotic cell death between the various cultures. The MSC culture under hypoxic conditions was associated with the induction of hypoxia-inducing factor-alpha (HIF-1α) and an elevated expression of energy metabolism-associated genes including GLUT-1, LDH and PDK1. Concomitantly, a significantly enhanced glucose consumption and a corresponding lactate production could be observed in the hypoxic MSC cultures suggesting an altered metabolism of these human stem cells within the hypoxic environment.
30 November 2014 in Applied Microbiology and Biotechnology
The growing world population, public awareness of animal welfare, environmental impacts and changes in meat consumption leads to the search for novel approaches to food production. Novel foods include products with a new or specifically modified molecular structure, foods made from microorganisms, fungi, algae or insects, as well as from animal cell or tissue cultures. The latter approach is known by various names: “clean meat”, “in vitro meat” and “cell‐cultured” or “(cell‐)cultivated meat”. Here, cells isolated from agronomically important species are expanded ex vivo to produce cell biomass used in unstructured meat or to grow and differentiate cells on scaffolds to produce structured meat analogues. Despite the fast‐growing field and high financial interest from investors and governments, cultivated meat production still faces challenges ranging from cell source choice, affordable expansion, use of cruelty‐free and food‐grade media, regulatory issues and consumer acceptance. This overview discusses the above challenges and possible solutions and strategies in the production of cultivated meat. The review integrates multifaceted historical, social, and technological insights of the field, and provides both an engaging comprehensive introduction for general interested and a robust perspective for experts.
Many properties in both healthy and pathological tissues are highly influenced by the mechanical properties of the extracellular matrix. Stiffness gradient hydrogels are frequently used for exploring these complex relationships in mechanobiology. In this study, the fabrication of a simple, cost‐efficient, and versatile system is reported for creation of stiffness gradients from photoactive hydrogels like gelatin‐methacryloyl (GelMA). The setup includes syringe pumps for gradient generation and a 3D printed microfluidic device for homogenous mixing of GelMA precursors with different crosslinker concentration. The stiffness gradient is investigated by using rheology. A co‐culture consisting of human adipose tissue‐derived mesenchymal stem cells (hAD‐MSCs) and human umbilical cord vein endothelial cells (HUVECs) is encapsulated in the gradient construct. It is possible to locate the stiffness ranges at which the studied cells displayed specific spreading morphology and migration rates. With the help of the described system, variable mechanical gradient constructs can be created and optimal 3D cell culture conditions can be experientially identified.