Integrated generation of induced pluripotent stem cells in a low-cost device.

Human induced pluripotent stem cells (iPSCs) have unlimited proliferation capability and potential to differentiate into all somatic cells. Their derivatives contain patients' genetic information and can model many diseases. Additionally, derivatives of patient-specific iPSCs induce minimal immune rejection in vivo. With this unique combination of properties, iPSCs open the avenue to personalized medicine including personalized drug screening, toxicity test, cell therapy and tissue engineering. However, the further advance of iPSC-based personalized medicine is currently limited by the difficulty to generate iPSCs for large populations and at affordable cost. We here report a low-cost device to address this challenge. The device allows the entire bioprocess for generating high quality and quantity of iPSCs for one patient to be done automatically within a closed conical tube without cell passaging. Additionally, iPSCs can be further differentiated into somatic cells in the device. Thus, the device also allows integrated iPSCs generation, expansion and differentiation to produce any somatic cell types. This device can be made in large quantities at low cost for manufacturing iPSCs (and their derivatives in necessary) for large populations at affordable cost. It will significantly advance the iPSCs-based personalized medicine.

Scalable and physiologically relevant microenvironments for human pluripotent stem cell expansion and differentiation.

Human pluripotent stem cells (hPSCs) are required in large numbers for various biomedical applications. However, the scalable and cost-effective culturing of high quality hPSCs and their derivatives remains very challenging. Here, we report a novel and physiologically relevant 3D culture system (called the AlgTube cell culture system) for hPSC expansion and differentiation. With this system, cells are processed into and cultured in microscale alginate hydrogel tubes that are suspended in the cell culture medium in a culture vessel. The hydrogel tubes protect cells from hydrodynamic stresses in the culture vessel and limit the cell mass smaller than 400 µm in diameter to ensure efficient mass transport, creating cell-friendly microenvironments for growing cells. This system is simple, scalable, highly efficient, defined and compatible with the current good manufacturing practices. Under optimized culture conditions, the AlgTubes enabled long-term culture of hPSCs (>10 passages, >50 days) with high cell viability, high growth rate (1000-fold expansion over 10 days per passage), high purity (>95% Oct4+) and high yield (5.0 × 108 cells ml-1), all of which offer considerable advantages compared to current approaches. Moreover, the AlgTubes enabled directed differentiation of hPSCs into various tissue cells. This system can be readily scaled to support research from basic biological study to clinical development and the future industry-scale production.

Manufacturing human pluripotent stem cell derived endothelial cells in scalable and cell-friendly microenvironments.

Human pluripotent stem cell derived endothelial cells (hPSC-ECs) are of great value for studying and treating vascular diseases. However, manufacturing high quantity and quality hPSC-ECs with current cell culture technologies remains very challenging. Here, we report a novel method that can manufacture hPSC-ECs in scalable and cell-friendly microenvironments to address this challenge. Using this method, hPSCs are expanded and differentiated into ECs in microscale alginate hydrogel tubes. The hydrogel tubes protect cells from the highly variable hydrodynamic conditions and critical hydrodynamic stresses in the culture vessel and limit the cell mass less than the diffusion limits (of human tissue) to ensure efficient mass transport. The hydrogel tubes provide uniform and friendly microenvironments for cells to grow. This novel design leads to extremely high production efficiency. We showed that hPSC-ECs could be produced in 10 days with high viability (>90%), high purity (>80%) and high yield (∼5.0 × 108 cells per mL of microspace). The yield is about 250 times that of the current-state-of-the-art. hPSC-ECs made in these hydrogel tubes had similar in vitro and in vivo functions to hPSC-ECs generated by conventional cell culture methods. This simple, scalable, efficient, defined and cost-effective technology will make hPSC-ECs broadly available and affordable for various biomedical applications.

A totally recombinant fibrin matrix for mesenchymal stem cell culture and delivery.

Mesenchymal stem cells (MSCs) have been widely studied for tissue engineering and treating diseases in laboratories, clinical trials, and clinics. Fibrin matrices are often used to culture MSCs or increase the retention of MSCs at the injection site. However, fibrins made with the human plasma derived fibrinogen have high cost and risk of human pathogen transmission. In this article, we studied if fibrin matrices made with recombinant human fibrinogen, recombinant human thrombin, and recombinant human factor XIII could be used to culture and deliver MSCs. We systematically investigated the relationships between the fibrin matrix formulation, its nanostructure, and the behaviors of the cells in the matrix including the cell morphology, viability, and growth. We found that the fibrinogen concentration significantly affected the matrix structure and cell behaviors. We then used an optimized fibrin matrix to deliver human MSCs into mice subcutaneously. We found that the matrix could significantly enhance the retention of MSCs at the injection site. To our best knowledge, this is the first study on using fibrin matrices made with entirely recombinant proteins for culturing and delivering MSCs. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 3135-3142, 2018.

Differentiating human pluripotent stem cells into vascular smooth muscle cells in three dimensional thermoreversible hydrogels.

Vascular smooth muscle cells (VSMCs) are of great value and are needed in large quantities for tissue engineering, drug screening, disease modeling and cell-based therapies. However, getting high quantity VSMCs remains a challenge. Here, we report a method for the scalable manufacturing of VSMCs from human pluripotent stem cells (hPSCs). hPSCs are expanded and differentiated into VSMCs in a three dimensional (3D) thermoreversible hydrogel. The hydrogel not only acts as a 3D scaffold for cells to grow, but also protects cells from hydrodynamic stresses in the culture vessel and prevents cells from excessive aggregation. Together, the hydrogel creates a cell-friendly microenvironment, leading to high culture efficiency. We show that VSMCs can be generated in 10 days with high viability (>90%), high purity (>80%) and high yield (∼2.0 × 107 cells per mL hydrogel) in the hydrogel scaffold. The generated VSMCs have normal functions. Genome-wide gene expression analysis shows VSMCs made in the hydrogel (i.e. 3D-VSMCs) have higher expression of genes related to vasculature development and glycolysis compared to VSMCs made in the conventional 2D cultures (i.e. 2D-VSMCs), while 2D-VSMCs have higher expression of genes related to cell proliferation. This simple, defined and efficient method is scalable for manufacturing hPSC-VSMCs for various biomedical applications.

Three-dimensional tissues using human pluripotent stem cell spheroids as biofabrication building blocks.

A recently emerged approach for tissue engineering is to biofabricate tissues using cellular spheroids as building blocks. Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), can be cultured to generate large numbers of cells and can presumably be differentiated into all the cell types of the human body in vitro, thus are an ideal cell source for biofabrication. We previously developed a hydrogel-based cell culture system that can economically produce large numbers of hPSC spheroids. With hPSCs and this culture system, there are two potential methods to biofabricate a desired tissue. In Method 1, hPSC spheroids are first utilized to biofabricate an hPSC tissue that is subsequently differentiated into the desired tissue. In Method 2, hPSC spheroids are first converted into tissue spheroids in the hydrogel-based culture system and the tissue spheroids are then utilized to biofabricate the desired tissue. In this paper, we systematically measured the fusion rates of hPSC spheroids without and with differentiation toward cortical and midbrain dopaminergic neurons and found spheroids' fusion rates dropped sharply as differentiation progressed. We found Method 1 was appropriate for biofabricating neural tissues.