Clinically amendable, defined, and rapid induction of human brain organoids from induced pluripotent stem cells

Human brain organoids provide opportunities to produce three-dimensional (3D) brain-like tissues for biomedical research and translational drug discovery, toxicology, and tissue replacement. Here we describe a protocol for rapid and defined induction of brain organoids from human induced pluripotent stem cells (iPSCs), using commercially available culture and differentiation media and a cheap, easy to handle and clinically approved semisynthetic hydrogel. Importantly, the methodology is uncomplicated, well-defined, and reliable for reproducible and scalable organoid generation, and amendable to principles of current good laboratory practice (cGLP), with the potential for prospective adaptation to current good manufacturing practice (cGMP) toward clinical compliance

Cell Processing for 3D Bioprinting: Quality Requirements for Quality Assurance in Fundamental Research and Translation

Bioprinting is an additive manufacturing process where biomaterials-based inks are printed layer-by-layer to create three-dimensional (3D) structures that mimic natural tissues. Quality assurance for 3D bioprinting is paramount to undertaking fundamental research and preclinical and clinical product development. It forms part of quality management and is vital to reproducible and safe tissue fabrication, function, and regulatory approval for translational application. This chapter seeks to place the implementation of quality practices in 3D bioprinting front-of-mind, with emphasis on cell processing, although important to all components and procedures of the printing pipeline

Culturing and Cryobanking Human Neural Stem Cells

The discovery and study of human neural stem cells has advanced our understanding of human neurogenesis, and the development of novel therapeutics based on neural cell replacement. Here, we describe methods to culture and cryopreserve human neural stem cells (hNSCs) for expansion and banking. Importantly, the protocols ensure that the multipotency of hNSCs is preserved to enable differentiation to neurons and supporting neuroglia

3D Bioprinting Electrically Conductive Bioink with Human Neural Stem Cells for Human Neural Tissues

Bioprinting cells with an electrically conductive bioink provides an opportunity to produce three-dimensional (3D) cell-laden constructs with the option of electrically stimulating cells in situ during and after tissue development. We and others have demonstrated the use of electrical stimulation (ES) to influence cell behavior and function for a more biomimetic approach to tissue engineering. Here, we detail a previously published method for 3D printing an electrically conductive bioink with human neural stem cells (hNSCs) that are subsequently differentiated. The differentiated tissue constructs comprise functional neurons and supporting neuroglia and are amenable to ES for the purposeful modulation of neural activity. Importantly, the method could be adapted to fabricate and stimulate neural and nonneural tissues from other cell types, with the potential to be applied for both research- and clinical-product development

3D Bioprinting Human Induced Pluripotent Stem Cell Constructs for In Situ Cell Proliferation and Successive Multilineage Differentiation

The ability to create 3D tissues from induced pluripotent stem cells (iPSCs) is poised to revolutionize stem cell research and regenerative medicine, including individualized, patient-specific stem cell-based treatments. There are, however, few examples of tissue engineering using iPSCs. Their culture and differentiation is predominantly planar for monolayer cell support or induction of self-organizing embryoids (EBs) and organoids. Bioprinting iPSCs with advanced biomaterials promises to augment efforts to develop 3D tissues, ideally comprising direct-write printing of cells for encapsulation, proliferation, and differentiation. Here, such a method, employing a clinically amenable polysaccharide-based bioink, is described as the first example of bioprinting human iPSCs for in situ expansion and sequential differentiation. Specifically, There are extrusion printed the bioink including iPSCs, alginate (Al; 5% weight/volume [w/v]), carboxymethyl-chitosan (5% w/v), and agarose (Ag; 1.5% w/v), crosslinked the bioink in calcium chloride for a stable and porous construct, proliferated the iPSCs within the construct and differentiated the same iPSCs into either EBs comprising cells of three germ lineages-endoderm, ectoderm, and mesoderm, or more homogeneous neural tissues containing functional migrating neurons and neuroglia. This defined, scalable, and versatile platform is envisaged being useful in iPSC research and translation for pharmaceuticals development and regenerative medicine

Human stem cells for modeling neurological disorders: accelerating the drug discovery pipeline

The availability of human stem cells heralds a new era for modeling normal and pathologic tissues and developing therapeutics. For example, the in vitro recapitulation of normal and aberrant neurogenesis holds significant promise as a tool for de novo modeling of neurodevelopmental and neurodegenerative diseases. Translational applications include deciphering brain development, function, pathologies, traditional medications, and drug discovery for novel pharmacotherapeutics. For the latter, human stem cell-based assays represent a physiologically relevant and high-throughput means to assess toxicity and other undesirable effects early in the drug development pipeline, avoiding late-stage attrition whilst expediting proof-of-concept of genuine drug candidates. Here we consider the potential of human embryonic, adult, and induced pluripotent stem cells for studying neurological disorders and preclinical drug development

Cryobanking Pluripotent Stem Cells

Cryobanking human pluripotent stem cells (hPSCs), be they human embryonic (hESCs) or induced pluripotent stem cells (iPSCs), is essential for their use in research and cell-based therapeutics. Working and master cell banks can be generated with a desired level of quality assurance applied during cell freezing and storage. Conventional vitrification has evolved to more advanced control rate freezing, culminating in a myriad of published protocols with variable proficiencies and clinical efficacies. Notwithstanding, standardized and reliable protocols are necessary for basic science through to applied research and clinical product development. This chapter details several methods for hPSC cryopreservation, suitable for routine application, high-quality research, and adaptable for clinical compliance

Smart graphene-cellulose paper for 2D or 3D origami-inspired human stem cell support and differentiation

Graphene-based materials represent advanced platforms for tissue engineering and implantable medical devices. From a clinical standpoint, it is essential that these materials are produced using non-toxic and non-hazardous methods, and have predictable properties and reliable performance under variable physiological conditions; especially when used with a cellular component. Here we describe such a biomaterial, namely smart graphene-cellulose (G-C) paper, and its suitability for traditional planar two-dimensional (2D) or three-dimensional (3D) human cell support, verified by adipose-derived stem cell (ADSC) culture and osteogenic differentiation. G-C paper is prepared using commercially available cellulose tissue paper as a substrate that is coated by immersion-deposition with graphene oxide (GO) followed by reduction to reduced graphene oxide (RGO) without the use of toxic organic solvents. The fabrication process is amenable to large scale production and the resultant papers have low electrical resistivity (up to ∼300 Ω/sq). Importantly, G-C papers can be configured to 3D constructs by lamination with alginate and further modified by folding and rolling for 3D origami-inspired cell-laden structures