Background & Aim Scalable and efficient expansion of cell cultures are an important need for pluripotent stem cell (PSC)-derived allogeneic cell therapies. The large-scale production of high-quality PSCs can be achieved by three-dimensional (3D) suspension culture, wherein PSCs are cultured as aggregates or spheroids. However, adoption of 3D PSC suspension culture in clinical workflows is limited by the lack of commercial options for PSC suspension culture media. To address this, we developed the new GMP manufactured Gibco™ Cell Therapy Systems (CTS) StemScale™ PSC Suspension Medium to support PSC-based clinical manufacturing workflows. CTS StemScale is xeno-free and enables single cells to self-aggregate into 3D spheroids for efficient cell expansion. Methods, Results & Conclusion CTS StemScale supports both induced pluripotent stem cells (iPSCs) and embryonic stem cells (ESCs), with cell line-dependent growth in the range of 5X – 10X expansion per passage. When cultured over multiple consecutive passages, these spheroids have been demonstrated to maintain pluripotency, genomic stability, and trilineage differentiation potential. This suspension culture approach enables easy scale-up in various cell culture vessel sizes, from small-scale (<100 mL) culture vessels to large-scale (>1L) culture systems inclusive of bioreactors. Notably, 450 million cells were expanded to 5 billion cells in 5 days by using this PSC culture medium to grow spheroids within a bioreactor. To better enable future scale-up or other downstream applications, we also cryopreserved these cells at high densities which would minimize the number of cryovials required to thaw. Cells thawed from these vials showed high viability and were able to form spheroids which were capable of expanding at normal rates. Ultimately, cells grown in CTS StemScale have the flexibility to differentiate as 3D spheroids, dissociate into single cells and be utilized in downstream applications, or be cryopreserved as single cells for future use.

Background & AimThe cell therapy field has grown exponentially as a prominent therapeutic for several diseases, including cancer. With growing interest in off-the-shelf autologous immune cell therapies, significant obstacles must still be overcome, including the ability to obtain large yields of the desired cell type. Pluripotent stem cells (PSCs) are a renewable and easily scalable cell source that could be used to generate large quantities of a given cell type for autologous cell therapies. Natural killer (NK) cells are an innate, cytotoxic lymphoid immune cell that can kill malignant cells without the need of HLA matching. Published NK cell therapy clinical trials utilized ∼5 × 106 to 1 × 108 total NK cells per kilogram body weight yet methods such as donor sourcing and in vitro PSC differentiation have long protocols requiring co-cultures and/or an embryoid body formation step, which hamper the ability to efficiently generate large quantities of functional NK cells. To overcome these drawbacks, we have developed a 3D PSC differentiation protocol using CTS-StemScale™ PSC suspension culture medium, which is compatible with clinical manufacturing workflows.Methods, Results & ConclusionWe focused on the scale up generation of hematopoietic stem cells (HSCs), a multipotent stem cell capable of generating NK cells, using the established StemPro-34 SFM media system supplemented with a unique combination of growth factors and cytokines. HSCs were further differentiated in static, or constant aggitation, to NK cells in the absence of exogenous feeder cells and subsequently expanded using CTS™ NK Xpander. Our iNK differentiation protocol has been evaluated in vessels up to a 100 mL mini bioreactor, producing ∼107 CD56+CD3- iNK cells. The iNKs produced in the protocol successfully kill immortalized cancer cell lines, as well as patient derived tumoroid lines. In summary, this iNK differentiation protocol shows promising scalability, and produced functional cytotoxic CD56+CD16+ without the use of feeder cells.

Umbilical-cord blood (UCB) is an important source of hematopoietic stem and progenitor cells (HSPC) for transplantation into patients lacking a suitable HLA-matched donor. However, due to the limited cell dose in each cord blood unit, individuals >60 kg are restricted from the use of UCB-based therapy. Ex vivo expansion of UCB CD34+ cells is one strategy employed to increase the hematopoietic cell dosage. A major limitation of current systems used for the expansion of HSPC is that ex vivo culture leads to expansion and differentiation at the expense of the most primitive pluripotent long-term stem cells. This has limited the clinical application of ex vivo expanded HSPC, since short-term progenitor cells only provide transient protection, ultimately reducing the long-term positive health outcomes, increasing the duration of hospitalizations, and health care costs per patient. Development of a culture system that expands both short-term and long-term HSPC would facilitate immune protection during the early phase of recovery, and provides a suitable solution for transfusion-independent hematopoiesis. Therefore, we sought to develop a HSPC culture medium that enables the expansion of both long-term and short-term HSPC, while maintaining their functional properties. To this end, we conducted several iterative rounds of Design of Experiments (DOE) involving multifactorial analysis, and mathematical modeling methods. Definitive Screening DOEs allowed us to identify optimal combinations and concentrations of essential media components, small molecules, and growth factors. The performance of candidate HSPC expansion media were evaluated after 7 days of culture, with the following attributes assessed: (1) viability of cells; (2) numbers of total nucleated cells; (3) percentages and numbers of CD34+ cells; (4) percentages and numbers of CD34+CD90+CD45RA- cells; (5) expression of aldehyde dehydrogenase by expanded CD34+ cells; and (6) colony-forming unit (CFU) assays. As the transplantation of HSPC in immuno-deficient mice is the gold standard in determining whether the expanded cells are engraftable, we plan to conduct these studies with the lead candidate HSPC expansion medium. Taken together, we seek to highlight our design philosophy in HSPC culture media development. We believe that our efforts are critical for the successful utilization of hematopoietic stem cell transplants in translational cell therapies.

Pluripotent stem cell (PSC) culture using the xeno-free Essential 8™ Medium/truncated recombinant human Vitronectin system has been shown to support normal PSC properties and provide a large pool of cells for disease modeling and drug development. As research moves from translational to clinical research, general regulatory guidance from the US Food and Drug Administration (FDA) indicates that, cGMP manufactured, or clinical grade reagents should be used whenever available as ancillary reagents to minimize downstream risk to patients. Thus, we sought to identify regulatory compliant, animal-origin-free alternatives for growth factors contained within the Essential 8™ Medium, producing a qualified ancillary system for PSC expansion. Here we present data to support a seamless transition from the xeno-free Essential 8™ Medium system to the Cell Therapy Systems (CTS™) animal-origin free system. Compatibility is shown with existing cGMP-manufactured passaging reagents: Versene Solution for clumped cell passaging and CTS™ TrypLE™ Select combined with RevitaCell™ Supplement for single cell passaging. Upon expansion, PSCs are shown to maintain normal PSC properties, including morphology, pluripotency, karyotype, and trilineage differentiation potential. Additional information on applicability of scale-up in adherent culture using the Nunc™ EasYDish™ Dishes and Nunc™ EasyFill™ Cell Factories is also provided. Together this system provides a consistent, feeder-free PSC culture medium for translational and clinical research.

Normal wound healing is characterized by a sequence of partially overlapping stages including inflammation, proliferation, and remodeling. When this sequence of events is perturbed, for instance by hypoxia, neuropathy or dysfunctional immune response, there is a risk of the wounds becoming chronic. Since chronic wounds represent a significant burden to both patients and health systems worldwide, there is significant interest in developing new treatment modalities. The ideal treatment would facilitate a nomalization of the wound healing process. As adipose-derived stem cells (ASCs) have pro-angiogenic, immunomodulatory and anti-apoptotic properties, they are proposed as candidates for regenerative therapies of chronic wounds. In order for successful treatments to be developed, it would be beneficial to have simple in vitro assays for each of the stages of wound healing. In particular, for the study of the proliferation stage of wound healing, the so-called scratch assay is often employed, as it mimics fibroblast and/or keratinocyte behavior. Here we describe modifications of this scratch assay in order to assess the effects of ASC secretome on fibroblast and keratinocyte migration and proliferation. Because optimal culture conditions for ASCs are different than for skin cells, particularly keratinocytes, assay modifications were required in order to evaluate stem cell derived factors in the scratch wounds. These included concentration of soluble factors by filtrations, media exchange by dialysis as well as evaluating culture parameters including seeding densities, feed schedules and surface coatings. Here we describe and quantify how these changes impact cell behavior in scratch wound assays using time-lapse imaging and analysis of scratch wound closure. We describe some of the pitfalls we experienced during this study and suggest possible solutions to make the fibroblast- or keratinocyte-based scratch assays suitable models to study ASC effects on wound healing.

Applications for induced pluripotent stem cells are extensive and include basic biology, drug discovery, toxicological assessment, tissue regeneration, and cancer therapy, among many others. However, reprogramming of somatic cells is an intensive and time consuming process and karyotypic abnormalities are known to occur after extended periods in culture. Therefore, the ability to cryopreserve iPSCs with a high survival rate would be advantageous. Although stem cells are most frequently cryopreserved as small cell clumps in suspension, colony dissociation is known to cause cell loss and increased differentiation. Thus cryopreservation in the adherent state would alleviate any need to dissociate stem cells from the culture surface and prevent any damage that is a result of dissociation. Vitrification methods are particularly promising for cryopreservation of adherent cells because damage due to extracellular ice is prevented. However, vitrification methods require the use of high CPA concentrations which increase the risk of osmotic and toxic damage. Recently, we developed a rational design algorithm for designing toxicity-minimized CPA addition and removal procedures. (J.D. Benson, A.J. Kearsley, A.Z. Higgins, Cryobiology 64 (2012) 144–151) To successfully apply these predicted procedures, accurate knowledge of cell biophysical parameters is required. The purpose of this study was to determine the necessary biophysical parameters for the rational design of CPA addition and removal procedures for vitrification of iPSCs. To determine membrane permeability parameters for human iPSCs, we adapted our calcein fluorescence quenching method (A.K. Fry, A.Z. Higgins, Cellular and Molecular Bioengineering 5 (2012) 287–298) for use with an automated plate reader. Permeability parameters were determined for dimethyl sulphoxide, ethylene glycol, glycerol, and propylene glycol at 4 °C, 21 °C, and 37 °C. Most notably, glycerol permeation was significantly slower than the other CPA types. To determine the osmotic tolerance limits of iPSCs, cells were exposed to test solutions, which included varying concentrations of hypotonic buffer and hypertonic sucrose, for 15 mins. Cell yield was assessed 24 h after cells were returned to culture using PrestoBlue®. Osmotic tolerance limits were determined for single cell suspensions and adherent iPSCs at 4 °C, 21 °C, and 37 °C. Using ANOVA analysis, the effect of the osmolality and temperature as well as the cross interaction between osmolality and temperature were significant for both hypotonic and hypertonic exposures. Also, the tolerable limits varied greatly depending on the test temperature. In particular, excessive cell volume changes were more damaging at 37 °C than 4 °C or 21 °C. The permeability parameters and osmotic tolerance limits presented in this study enable rational design of CPA addition and removal procedures. The information in this study is an important step toward development of successful vitrification strategies for adherent human iPSCs. Source of funding: This project was funded by a Collaborative Research Contract from Life Technologies with Oregon State University. Conflict of interest: None declared. adam.higgins@oregonstate.edu

Specific gene ablation by RNA inference (RNAi) involves the binding of short interfering RNA (siRNA), 21 to 22 nucleotides long, to complementary mRNA sequences, leading to sequence-specific posttranslational gene silencing, thus providing a powerful tool for studying gene function with potential therapeutic applications. Here we describe the development of a two-vector adenovirus system for efficient, tightly controlled hairpin siRNA expression (shRNA). Regulated expression of the shRNA is conferred within an adenoviral vector by a modified RNA polymerase III promoter containing a Tet operator element adjacent to the transcription start site. In the presence of the tetracycline repressor protein (TetR), encoded in a second adenovirus, shRNA expression is repressed. Addition of tetracycline abolishes TetR binding, allowing shRNA transcription to proceed, and leading to reduced mRNA and protein expression. Here we establish the efficacy of this system by delivering siRNA targeted against the transcriptional coactivator p300. Our results show tetracycline-mediated inhibition of p300 mRNA and protein accumulation in the presence of both viruses, but no effect in the absence of antibiotic. Regulated adenoviral shRNA vectors offer the advantages of being able to infect a wide array of replicating and nonreplicating cells and of allowing temporal control of gene silencing.

Peptide growth factors regulate cell fate by activating distinct signal transduction pathways that ultimately influence gene expression. Insulin-like growth factors (IGFs) play central roles in controlling somatic growth and participate in skeletal muscle development and regeneration. In cultured muscle cells, IGF action is critical both for maintaining viability during the transition from proliferating to differentiating myoblasts and for facilitating differentiation. By contrast, platelet-derived growth factor (PDGF) can sustain cell survival but inhibits differentiation. Here we examine the genetic programs that accompany IGF and PDGF action in myoblasts. Through analysis of high-density oligonucleotide arrays containing approximately 36,000 mouse probe sets, we identify 90 transcripts differentially induced by IGF-I, including 28 muscle-specific genes and 33 previously unannotated mRNAs, and 55 transcripts specifically stimulated by PDGF, including 14 unknowns. Detailed study of one IGF-induced mRNA shows that it encodes a protein related to a recently characterized repulsive guidance molecule postulated to regulate neuronal targeting during development. Our results demonstrate the power of transcriptional profiling for gene discovery and provide opportunities for investigating new proteins potentially involved in different aspects of growth factor action in muscle.

IL-10 plays an important role in preventing excessive inflammation to the normal flora in the intestinal lumen. The purpose of this study was to compare the effect of normal flora on inflammation in mice in which the IL-10 gene was disrupted. IL-10 knock-out mice housed in germfree conditions remained healthy while those housed in conventional conditions developed colitis after weaning, suggesting that IL-10 inhibits the adverse responses to luminal Ag. Crypt abscesses were present in virtually all of the diseased animals as evidenced by flattening of the epithelial cells and a large number of neutrophils in the lumen of the crypt. Since KC is a chemokine that is capable of recruiting neutrophils in mice, mRNA and protein for KC was measured. Increased levels of both KC mRNA and protein were detected in the colon of diseased mice. To determine whether the epithelial cells were capable of synthesizing KC and contributing to neutrophil accumulation in the crypts, a murine intestinal epithelial cell line (Mode-K) was shown to express mRNA and protein for KC. Two cytokines induced in association with colitis in these mice, TNF-alpha and IFN-gamma, increased the expression of KC mRNA and protein in murine epithelial cells. However, IL-10 was incapable of decreasing the induction of KC, even though the cells expressed the IL-10 receptor. These results suggest that the neutrophil chemokine KC is produced by gastrointestinal epithelial cells in response to inflammatory mediators that are expressed following exposure to normal flora in animals lacking IL-10.