Genome editing —precise, site-specific DNA modification —can now be achieved through the use of chimeric protein constructs that consist of a sequence-specific binding protein linked to a non-specific endonuclease that cleaves DNA a predictable distance from the binding site. The DNA-binding domains of transcription activator–like (TAL) effectors are known and programmable, and that knowledge can be used to create customized proteins that bind specifically to virtually any desired DNA sequence. Recently, clustered regulatory interspaced short palindromic repeats (CRISPRs), together with CRISPR-associated (Cas) endonucleases, have also been used for genomic editing. Like the chimeric TAL effector nucleases (TALENs), these RNA-guided endonuclease (RGEN) systems also have modular DNA recognition and cleavage functions—by engineering the DNA-recognition components, the endonuclease components of CRISPR/Cas systems can be targeted with high specificity to cut any genomic sequence desired.
How does genome editing fit into your research, now and in the future? This Ask the Expert session covered topics including, deciding which technology to use for specific research applications, including cell type, desired modification, target sequence constraint’s etc.
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.
During this Ask the Experts session, we discussed the challenges associated with hPSC differentiation to neural and cardiac lineages, how these processes can be efficiently simplified with tools and cGMP cell culture media systems for robust, efficient and scalable differentiation of these two critical cell lineages. Use of these reagent systems will enable researchers to precisely control and direct the differentiation to terminal lineages in a relatively easy manner, and speedily with high efficiency.
Cell Proliferation assays are an important set of fluorescence based tests that can monitor cell health, cell division, and cell proliferation using a variety of techniques involving flow cytometry and imaging platforms. From DNA content cell cycle, to tracking of generational cell division, to simple viability and vitality measurements, there are assays that can provide a rich data set to answer simple or complex questions and provide direction for future experimentation.
Are my cells alive? Are my cells dividing and proliferating? Are my cells healthy? Are you having issues with cell cycle measurements? In this ask-an-expert session, question topics included fluorescent testing measuring cell proliferation and assessing cell health using flow cytometry and imaging platforms.
Typically, DNA enters the nucleus when cells divide because cell division creates small nuclear pores. In non-dividing primary cells DNA doesn’t enter the nucleus making these cells very hard-to-transfect. If DNA entry is a bottleneck, why not deliver mRNA directly?
This Ask the Expert session included readers’ questions on transfection and topics included specific applications and troubleshooting. What’s the best way to transfect mRNA? Which reagent should I use? Can I transfect primary cells with a reagent? How do I prepare an mRNA template from DNA? What’s the protocol?
The vast advances in technologies for the efficient generation of footprint-free induced pluripotent stem cells (iPSC) have led to the creation of several iPSC lines from varying sources, genetic backgrounds, and derivations in different medias and growth conditions, thus necessitating thorough characterization of the resulting cell lines.
One critical step in establishing iPSC lines involves the early identification of true iPSC clones and their subsequent characterization to ensure functional pluripotency. Various methods of characterization, ranging from visual morphological observation to the use of differentially expressed biomarkers are utilized for the initial identification of pluripotent cells. Dyes such as Alkaline Phosphatase Live Stain enable the early detection of emerging iPSC colonies that can be used in combination with morphological assessment to pick the right iPSC clone for further expansion. Established clones are further subjected to a combination of in vitro and in vivo cellular analysis to confirm functional pluripotency based on expression of self-renewal markers and trilineage differentiation potential. While such traditional methods have been successfully used, there is a need for a uniform standardized method for comprehensive characterization. In this Ask the Expert session, various options for identification and characterization of pluripotent stem cells was discussed.
Adherent cell culture is often used in the manufacture of biologic products, including vaccines. Historically, the surface for cells to adhere to has been provided either by a two-dimensional (2D) system such as roller bottles, T-flasks, cell factories or cell stacks; or microcarrier beads within a traditional stirred tank bioreactor. However the need to increase product production and reduce manufacturing footprints have led to the creation of novel solutions.
One alternative solution to traditional 2D systems and to a microcarrier-stirred tank option is a fixed-bed disposable bioreactor, which can provide efficient scale-up and manufacturing with a small footprint. In this Ask the Expert session, readers submitted qusetions about the use of fixed bed bioreactors for adherent cell manufacturing.
The full realization of the therapeutic potential of stem cells has only recently come into the forefront of regenerative medicine. Promising in vivo results have fueled the enthusiasm among basic researchers and their clinical colleagues and thus have widened the scope of stem cell application in human disease but major scientific and regulatory challenges exist and must be addressed in order to both facilitate the “bench to bedside” process of this nascent technology as well as enhance safety of the final cell product. One potential key to advancing stem cell therapies is the use of plant-based biologics as a defined, recombinant alternative to animal sourced components. Human LIF protein (rhLIF) was expressed in rice grain using a plant-based expression platform and demonstrated a 97% purity of the protein. The rhLIF was then used in stem cells to promote cell proliferation and maintenance of the pluripotent state. Ask the Expert question topics included LIF itself, the application of LIF, or animal component-free stem cell media.
Microbial fermentation processes are used for biomanufacturing of various drugs and vaccines, such as hormones, antibody fragments, and pneumococcal vaccine. Stirred-tank fermentors up to 100,000 L scale have traditionally been used in such microbial processes and their success has formed the general engineering foundation and principles of the design of bioreactors. The majority of today’s fermentation processes are performed in bioreactors constructed of traditional materials such as stainless steel. However, there is an increased interest in disposable technology to gain flexibility, save batch change-over time, and minimize cleaning and cleaning validation efforts.
To date, single-use stirred-tank bioreactors for mammalian and insect cell cultures have been successfully used in scales up to 2,000 L working volume and are installed in both clinical and commercial drug manufacturing facilities. However, for bioreactors to be utilized in microbial fermentation some engineering challenges needed to be addressed. For instance, fermentors had to be designed to handle very high metabolic rates and the high oxygen demands of some microbial cultures. By applying general bioengineering principles and designs, high oxygen transfer rates can be achieved also in disposable fermentors. Augmented designs and operational methods compensate for the low heat transfer rates in these systems. Although pressurizable single-use stirred-tank fermentors are within the realm of the technical feasibility, this feature might not be necessary as sufficient oxygen transfer can be achieved through a variety of mechanical and process control designs and techniques. This Ask the Expert session, answered readers’ questions about implementing single-use for microbial fermentation and troubleshooting.
Cell culture media, including basal media and feeds, are key elements impacting the performance of bioprocesses. Advancements in cellular and metabolic understanding, coupled with high throughput applications, have led to evolved approaches in medium development and optimization resulting in innovative cell culture media with desired characteristics to meet specific needs.
However, challenges associated with the use of cell culture media still exist. Examples include manufacturability, subpar stability, inconsistent performance and/or underperformance in terms of productivity and quality attributes. In some cases, troubleshooting efforts can be guided by learned know-how but in other circumstances a systematic approach is necessary to identify the root cause. In this Ask the Expert session, question topics included cell culture media design and problem solving.
Induced pluripotent stem (iPS) cell-based models hold tremendous potential for the study of human neurological disease. Advances in technologies, including improvements in the ease and efficiency of generating neural progenitor cells from iPS cells, have resulted in increased adoption of these models by the neuroscience community.
There are many options available to researchers interested in developing iPS-based models to complement traditional methods of neuroscience research. Points of consideration include the type of somatic cell used to generate the iPS colonies, the reprogramming system, the conditions under which iPS cells are maintained, the protocols used for neural induction and terminal differentiation, and the end-point assays used to investigate disease phenotype. In this Ask the Expert session, readers asked several questions related to implementing the use of iPS cells in disease modeling.