I have compiled a list of our top ten Cell Culture Dish “Ask the Expert” Sessions for 2015. Here are the top sessions in alphabetical order.
Human induced pluripotent stem (iPS) cells have the potential to greatly impact many areas of research and medicine and can be generated by reprogramming somatic cells through transient overexpression of key reprogramming factors.
While dermal fibroblasts were the first human cell type to be reprogrammed into iPS cells, blood cells are also increasingly being utilized as a starting cell type due to the limited invasiveness of sample collection, and the availability of banked blood samples representing a variety of disease, age, gender and geographical subtypes.
The choice of cell type to use for reprogramming is based on:
- The accessibility of tissue samples
- The genetic make-up of the target cells
- The reprogramming efficiency
In this session we had many great questions and useful suggestions on issues including reprogramming methods, differentiation techniques, confirming pluripotency, and matrices. We also discussed advantages and challenges of utilizing blood cells over other cell sources.
Sometimes it’s good to get back to basics. This approach can be especially helpful in cell culture particularly in troubleshooting any culture problems. In this session we had many interesting questions and helpful suggestions on topics including avoiding contamination, cell growth and passaging and proper storage techniques. Specific questions involved troubleshooting contamination, the use of antibiotics, and problems with freezing and thawing cells. We also featured a tip of the day with useful cell culture tips and tricks.
The culture of hematopoietic stem and progenitor cells (HSPCs) is important to assay the quality and functional properties of HSPCs e.g., during the implementation of transplantation or other approaches to treat hematological disorders, such as leukemia. Examples of HSPC culture assays include colony-forming unit (CFU) assays in semi-solid methylcellulose-based media, such as MethoCult™, to identify and quantify HSPCs, and expansion cultures in liquid media, such as StemSpan™, to increase HSPC numbers or generate large numbers of mature blood cells. These culture methods can also used to evaluate the efficacy and toxicity of new drug candidates on hematopoiesis in vitro and to generate target cells for reprogramming to generate induced pluripotent stem cells.
During this Ask the Expert session, we discussed HSPC culture, including the selection and evaluation of media and its components, using small molecules for HSPC expansion and factors affecting cell doubling time. Other topics included research into culture methods to increase the successful engraftment of transplanted cells, generating iPSCs from cord blood and isolating hematopoietic stem cells.
The promise of human pluripotent stem cells will be realized only when these cells are successfully coaxed into different cell types found in the human body, through the process of directed differentiation. This is critical to getting the desired cell types and numbers needed for drug screening, translational cell therapy and regenerative medicine applications. Most of the existing methods of differentiation are suboptimal, involving laborious mechanical and manual steps leading to issues of reproducibility and reduced efficiency in downstream processing of functionally mature lineages. The complex developmental process of differentiation and the challenges associated need to be efficiently deciphered in order to successfully direct the hPSC differentiation to target cell types.
In this session we had several interesting questions and helpful tips on topics including challenges with reproducibility of differentiation protocols, maintaining differentiation potential after thaw, selection of cell culture surfaces and methods for confirming differentiation. Cell lines covered included neural stem cells, cardiomyocytes, hepatocytes, and blood cells.
Intestinal Organoids – An important advancement in the way we study intestinal and adult stem cell biology
Intestinal organoids provide a major advancement in the way we study intestinal and adult stem cell biology. When intestinal crypts containing LGR5+ intestinal stem cells (ISCs) are placed in a specialized medium that simulates the intestinal stem cell niche, the ISCs rapidly proliferate and differentiate into all of the major cell types found in the intestinal epithelium: Paneth cells, goblet cells, enteroendocrine cells, and enterocytes. Remarkably, these cells arrange themselves into a crypt-villus structure surrounding a functional central lumen, thus maintaining the stem cell pool while mimicking the physiology of the adult intestinal epithelium. The resulting organoids (or “mini-guts”) can be used for a variety of research applications including:
- intestinal stem cell biology
● intestinal disease
● adult stem cell biology
● gene therapy
● preclinical drug screening
● personalized medicine
As an in vitro system that is capable of providing in vivo insight, intestinal organoids signify an exciting new chapter for research in these fields.
In this session we had many great questions and useful suggestions on issues including culture medium, growth, passaging, culturing techniques, the use of antibiotics, and recommendations in matrix selection and vessels. We also discussed the impact to animal testing, drug development, and personalized medicine.
Enhancing protein production is a common goal in the biomanufacturing industry. During this Ask the Expert session, we discussed the challenges associated with animal origin free media optimization and how defined supplements can provide an increase in productivity and yield. Essential Pharmaceuticals is addressing the challenge of animal origin free media optimization with their Cell-Ess supplement. Use of this product at 1% concentration resulted in a 37% increase in productivity. When using the supplement as a feed it resulted in in a 25% increase in yield and an extension of peak protein production with functional protein increase and desired glycosylation achieved. Results suggest that an increase in protein production may not necessarily require a change in the metabolic state of the cells.
In this discussion, specific culture related questions included cell types tried, media tested, benefits to already high titer cultures, impact to metabolic profile, peak protein yield, effect on downstream purification, and use in vaccine manufacturing. There were also manufacturing focused questions related to cost analysis, specific productivity, glycosylation profile, and regulatory considerations.
Optimization of serum free media can be a complex and daunting challenge for scientists working to bring a cellular therapy to market. When considering raw material sourcing, it is important to limit animal or human serum derived components as these are major sources of variability and potential viral contamination risks. Inclusion of recombinant versions represents a viable alternative to circumvent issues associated with serum-derived proteins. However, utilization of these components can sometimes bring new hurdles to light.
During this Ask the Expert session, we discussed optimizing primary and stem cell culture to reduce or remove serum. Topics covered included resolving variability issues, reducing versus eliminating serum, media screening, animal free vs. serum free ad coating matrix.
Mammalian transient expression allows for the rapid generation, purification, and characterization of milligram to gram quantities of secreted or intracellular recombinant proteins for therapeutic, functional, and structural studies.
In this session we had several interesting questions and helpful tips on topics including best practices, optimal complexation, best cell densities, achieving high volumes and expressing intercellular proteins. We also covered an overview of transient systems, transient expression media, and increasing expression yields.
Live cell fluorescence imaging provides the opportunity to study cellular function, however there can be challenges with the ability to image weak fluorophors without damaging the health of your cells.
In this session we had several interesting questions focused on combating the negative effects of imaging on cell health and viability including looking at media options as a possible solution. Other topics included vessels for imaging, fluorescent fusion proteins and options for evaluating cell viability.
CHO cells are the predominant host for biotherapeutic protein expression, with roughly 70% of licensed biologics manufactured in CHO. Multiple attributes make CHO cells desirable for bioproduction including the ability to adapt to high-density suspension culture in serum-free and chemically-defined media and the incorporation of post-translational modifications that are biologically-active in humans. For these reasons, the ability to produce transient CHO-derived proteins early on during drug development is highly advantageous to minimize, as much as possible, changes in protein quality/function observed when moving from R&D to bioproduction. Unfortunately, CHO cells express lower levels of protein than HEK293 cells in existing transient systems, in some instances 50-100 times less than the best 293-based systems. The recent introduction of the ExpiCHO transient expression system allows for unprecedented access to CHO-derived proteins early on during candidate selection and may serve to revolutionize the use of CHO cells for transient protein expression during the drug development process.
During this Ask the Expert session, we discussed the advantages of a utilizing transient CHO system for protein production. Topics covered included expected yield, post translational modifications, consistency with stable cell lines, comparison to 293 transient systems. Specific cell culture related questions included, how to optimize transfection and some of the regularly mentioned strategies, i.e. temperature shifts, cell densities, growth factors, and DNA concentration.