A scalable and physiologically relevant system for human induced pluripotent stem cell expansion and differentiation

Human induced pluripotent stem cells (iPSCs) and their derivatives are needed in large numbers for various biomedical applications. However, scalable and cost-effective manufacturing of high quality iPSCs and their derivatives remains a challenge. In vivo, cells reside in a 3D microenvironment that has plenty of cell-cell and cell-ECM (extracellular matrix) interactions, sufficient supply of nutrients and oxygen, and minimal hydrodynamic stresses. The current iPSC culturing methods, however, provide highly-stressed culturing microenvironments, leading to low culture efficiency. For instance, we and others showed iPSCs typically expanded 4-fold/4 days to yield ~2.0x10^6 cells/mL with current 3D suspension culturing. These cells occupy ~0.4% of the bioreactor volume. To our best knowledge, the largest culture volume demonstrated to date for iPSCs is less than 10 liters. There is a critical need to develop new culture technologies to achieve the iPSCs’ potential. Please click Additional Files below to see the full abstract

A simple and scalable hydrogel-based system for culturing protein-producing cells

Recombinant protein therapeutics have become important components of the modern medicine. Majority of them are produced with mammalian cells that are cultured either through adherent culturing, in which cells are cultured on substrates, or suspension culturing, in which cells are suspended and cultured in agitated cell culture medium in a culture vessel. The adherent cell culturing method is limited by its low yield. In suspension culturing, cells need extensive genetic manipulation to grow as single cells at high density, which is time and labor-consuming. Here, we report a new method, which utilizes a thermoreversible hydrogel as the scaffold for culturing protein-expressing cells. The hydrogel scaffolds not only provide 3D spaces for the cells, but also act as physical barriers to prevent excessive cellular agglomeration and protect cells from the hydrodynamic stresses. As a result, cells can grow at high viability, high growth rate, and extremely high yield even without genetic manipulations. The cell yield in the hydrogels is around 20 times of the suspension culturing. In addition, the protein productivity per cell per day in the hydrogel is higher than the adherent culturing method. This new method is simple, scalable and defined. It will be of great value for both the research laboratories and pharmaceutical industry for producing proteins

Scalable Production of Glioblastoma Tumor-initiating Cells in 3 Dimension Thermoreversible Hydrogels

There is growing interest in developing drugs that specifically target glioblastoma tumor-initiating cells (TICs). Current cell culture methods, however, cannot cost-effectively produce the large numbers of glioblastoma TICs required for drug discovery and development. In this paper we report a new method that encapsulates patient-derived primary glioblastoma TICs and grows them in 3 dimension thermoreversible hydrogels. Our method allows long-term culture (~50 days, 10 passages tested, accumulative ~\u3e1010-fold expansion) with both high growth rate (~20-fold expansion/7 days) and high volumetric yield (~2.0 × 107 cells/ml) without the loss of stemness. The scalable method can be used to produce sufficient, affordable glioblastoma TICs for drug discovery

Scalable Production of Glioblastoma Tumor-initiating Cells in 3 Dimension Thermoreversible Hydrogels

There is growing interest in developing drugs that specifically target glioblastoma tumor-initiating cells (TICs). Current cell culture methods, however, cannot cost-effectively produce the large numbers of glioblastoma TICs required for drug discovery and development. In this paper we report a new method that encapsulates patient-derived primary glioblastoma TICs and grows them in 3 dimension thermoreversible hydrogels. Our method allows long-term culture (~50 days, 10 passages tested, accumulative ~\u3e1010-fold expansion) with both high growth rate (~20-fold expansion/7 days) and high volumetric yield (~2.0 × 107 cells/ml) without the loss of stemness. The scalable method can be used to produce sufficient, affordable glioblastoma TICs for drug discovery

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

Human pluripotent stem cells (hPSCs), including human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs), are attractive cell sources for various biomedical applications including cell therapies, tissue biofabrication, drug screening and toxicity tests. These applications require large numbers of high quality cells. However, the scalable and cost-effective culturing of high quality hPSCs and their derivatives, especially for clinical applications, remains a challenge. In vivo, majority of human cells including the hESCs reside in 3D microenvironments that have plenty of cell-cell and cell-ECM (extracellular matrix) interactions, sufficient supply of nutrients, oxygen and growth factors, and no or minimal hydrodynamic stresses. The current hPSC culturing methods, however, provide culturing conditions that are very different from these physiological microenvironments, leading to low culture efficiency and difficulty to culture cells at large scales. For instance, we and others showed hPSCs typically expanded 4-fold in 4 days to yield around 2.0x10^6 cells/mL with current 3D suspension culturing. These cells occupy ~0.4% of the bioreactor volume. To our best knowledge, the largest 3D suspension culture volume demonstrated to date for hPSCs is less than 10 liters. Please click Additional Files below to see the full abstract

Comparative study of differentiating human pluripotent stem cells into vascular smooth muscle cells in hydrogel-based culture methods

Vascular smooth muscle cells (VSMCs), which provides structural integrity and regulates the diameter of vasculature, are of great potential for modeling vascular-associated diseases and tissue engineering. Here, we presented a detailed comparison of differentiating human pluripotent stem cells (hPSCs) into VSMCs (hPSCs-VSMCs) in four different culture methods, including 2-dimensional (2D) culture, 3-dimensional (3D) PNIPAAm-PEG hydrogel culture, 3-dimensional (3D) alginate hydrogel culture, and transferring 3- dimensional alginate hydrogel culture to 2-dimensional (2D) culture. Both hydrogel-based culture methods could mimic in vivo microenvironment to protect cells from shear force, and avoid cells agglomeration, resulting in the extremely high culture efficiency (e.g., high viability, high purity and high yield) compared with 2D culture. We demonstrated hPSC-VSMCs produced from hydrogel-based culture methods had better contractile phenotypes and the potential of vasculature formation. The transcriptome analysis showed the hPSC-VSMCs derived from hydrogel-based culture methods displayed more upregulated genes in vasculature development, angiogenesis and blood vessel development, extracellular matrix compared with 2D culture. Taken together, hPSC-VSMCs produced from hydrogel-based culture system could be applied in various biomedical fields, and further indicated the suitable development of alginate hydrogel for industrial production by taking all aspects into consideration

Fabricating 3-dimensional human brown adipose microtissues for transplantation studies

Transplanting cell cultured brown adipocytes (BAs) represents a promising approach to prevent and treat obesity (OB) and its associated metabolic disorders, including type 2 diabetes mellitus (T2DM). However, transplanted BAs have a very low survival rate in vivo. The enzymatic dissociation during the harvest of fully differentiated BAs also loses significant cells. There is a critical need for novel methods that can avoid cell death during cell preparation, transplantation, and in vivo. Here, we reported that preparing BAs as injectable microtissues could overcome the problem. We found that 3D culture promoted BA differentiation and UCP-1 expression, and the optimal initial cell aggregate size was 100 μm. The microtissues could be produced at large scales via 3D suspension assisted with a PEG hydrogel and could be cryopreserved. Fabricated microtissues could survive in vivo for long term. They alleviated body weight and fat gain and improved glucose tolerance and insulin sensitivity in high-fat diet (HFD)-induced OB and T2DM mice. Transplanted microtissues impacted multiple organs, secreted protein factors, and influenced the secretion of endogenous adipokines. To our best knowledge, this is the first report on fabricating human BA microtissues and showing their safety and efficacy in T2DM mice. The proposal of transplanting fabricated BA microtissues, the microtissue fabrication method, and the demonstration of efficacy in T2DM mice are all new. Our results show that engineered 3D human BA microtissues have considerable advantages in product scalability, storage, purity, safety, dosage, survival, and efficacy

Engineered Microenvironment for Manufacturing Human Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells

Human pluripotent stem cell-derived vascular smooth muscle cells (hPSC-VSMCs) are of great value for disease modeling, drug screening, cell therapies, and tissue engineering. However, producing a high quantity of hPSC-VSMCs with current cell culture technologies remains very challenging. Here, we report a scalable method for manufacturing hPSC-VSMCs in alginate hydrogel microtubes (i.e., AlgTubes), which protect cells from hydrodynamic stresses and limit cell mass to \u3c400 \u3eμm ensure efficient mass transport. The tubes provide cells a friendly microenvironment, leading to extremely high culture efficiency.We have shown that hPSC-VSMCs can be generated in 10 days with high viability, high purity, and high yield (~5.0 x 108 cells/mL). Phenotype and gene expression showed that VSMCs made in AlgTubes and VSMCs made in 2D cultures were similar overall. However, AlgTube-VSMCs had higher expression of genes related to vasculature development and angiogenesis, and 2D-VSMCs had higher expression of genes related to cell death and biosynthetic processes

A Scalable and Efficient Bioprocess for Manufacturing Human Pluripotent Stem Cell-Derived Endothelial Cells

Endothelial cells (ECs) are of great value for cell therapy, tissue engineering, and drug discovery. Obtaining high-quantity and -quality ECs remains very challenging. Here, we report a method for the scalable manufacturing of ECs from human pluripotent stem cells (hPSCs). hPSCs are expanded and differentiated into ECs in a 3D thermoreversible PNIPAAm-PEG hydrogel. The hydrogel protects cells from hydrodynamic stresses in the culture vessel and prevents cells from excessive agglomeration, leading to high-culture efficiency including high-viability (\u3e90%), high-purity (\u3e80%), and high-volumetric yield (2.0 x 107 cells/mL). These ECs (i.e., 3D-ECs) had similar properties as ECs made using 2D culture systems (i.e., 2D-ECs). Genome-wide gene expression analysis showed that 3D-ECs had higher expression of genes related to vasculature development, extracellular matrix, and glycolysis, while 2D-ECs had higher expression of genes related to cell proliferation

Scalable Culturing of Primary Human Glioblastoma Tumor- Initiating Cells with a Cell-Friendly Culture System

Glioblastoma is the most aggressive and deadly brain cancer. There is growing interest to develop drugs that specifically target to glioblastoma tumor-initiating cells (TICs). However, the cost-effective production of large numbers of high quality glioblastoma TICs for drug discovery with current cell culturing technologies remains very challenging. Here, we report a new method that cultures glioblastoma TICs in microscale alginate hydrogel tubes (or AlgTubes). The AlgTubes allowed long-term culturing (~50 days, 10 passages) of glioblastoma TICs with high growth rate (~700-fold expansion/14 days), high cell viability and high volumetric yield (~3.0 × 108 cells/mL) without losing the stem cell properties, all offered large advancements over current culturing methods. This method can be applied for the scalable production of glioblastoma TICs at affordable cost for drug discovery