43 Proteomics, Genomics & Microarrays Subculture


Stem cells undergo cellular division in the culture vessel. As they expand, they will eventually outgrow their home and must be subcultured to separate fl asks to provide space for further growth. Common practice is to use a digestive enzyme to free the stem cells from the culture surface. Trypsin isolated from bovine is commonplace in the tissue culture laboratory. Advances in the products available today use trypsin expressed in maize that is stable at room temperature in solution. Collagenase is an alternative dissociation reagent that is gentle and effi cient on a wide range of cells and is available both animal- free and GMP grade - again enabling robust consistent culture conditions, and removing the dependence on animal derived products that are inherently variable.


Cryopreservation


The stem cells harvested from cultures can be frozen and stored (or ‘cryopreserved’) safely for several decades. When required, the cryopreserved stem cells may be defrosted, revived and expanded in culture providing a renewable source of stem cells. During cryopreservation of stem cells, it is critical to prevent cell death and changes in genotype/phenotype. Today’s cryopreservation media can maintain consistent high cell viability after thawing; maintaining cell pluripotency, normal karyotype and proliferation even after long term cell storage. Traditionally, the cryopreservation process involved a rate-controlled freezer or a specialised container to freeze the cells at -1ºC/min. Advances in cryopreservation agents have removed the need for rate-controlled freezing. The process is now simple - you just place the stem cell suspension into a -80ºC freezer. Moreover, cryopreservation agents are available in GMP grade and with no animal-derived ingredients.


Differentiation


The power of stem cells lies in their ability both to self-renew and to differentiate into specialised cell types. The process of differentiation removes the stem cells from the workfl ow towards applications. Directed differentiation of stem cells into specifi c cell types enables the number of applications to grow. A typical differentiation protocol uses stepwise changes in culture medium, cytokines, growth factors and extracellular matrix over several weeks to direct the stem cells into a particular lineage and fate. Today, innovative technologies use genetic reprogramming factors that rapidly (< 1 week) differentiate stem cells into mature cell phenotypes. This advance signifi cantly reduces time to experiment and increases manufacturing capacity for differentiated cell types.


Table 1. Advances in Stem Cell Technologies. Description


Extracellular Matrix Culture Medium Growth Factors Dissociation Reagents


Figure 3. tem cell differentiated skeletal muscle myotubes disease model. Image courtesy of Jason D Doles, Rochester, Minnesota, USA.


Farming Meat in a Dish Area of Innovation


Recombinant Laminin Expressed in CHO and Silkworm


No medium change required over the weekend, GMP grade, animal free


Recombinant, GMP grade, animal free


Trypsin enzyme recombinantly expressed in maize. Collagenase & Neutral Protease expressed in Clostridium histolyticum


Cryopreservation Differentiation


Rate-controlled freezing not required. GMP grade, animal free and available for clinical use. Suitable for all cell types.


Rapid directed differentiation through genetic reprogramming


Examples of Innovative Products


iMatrix-511 StemFit Medium StemFit Purotein TrypLE


Collagenase NB Neutral Protease NB


STEM-CELLBANKER


Quick-Skeletal Muscle Quick-Endothelium Quick-Neuron


Future Technologies and Applications


Disease Modelling There are unlimited applications that arise from a renewable source of mature cell types. One exciting area of innovation using differentiated stem cells is in disease modelling. Studying a disease state in an organ or tissue has in the past been limited to using in vivo animal models; whereas, differentiated stem cells opened the opportunity to create disease states in specifi c cell types in vitro. In addition, current technologies enable organoids or ‘mini organs’ to be generated in the laboratory. Disease specifi c induced pluripotent stem cells can also be used to create disease models in vitro that are valuable tools for the study of disease and drug development without the need for in vivo animal models. In theory, any tissue is possible to create in vitro. In an exciting example of stem cell disease modelling, Dr Takayama from the CiRA in Kyoto, Japan has successfully modelled the life cycle of SARS-CoV-2 in both organoids and undifferentiated pluripotent stem cells (Takayama, 2020) (Sano, 2021) (Figure 2). In another example, the Skeletal Muscle Differentiation Kit was used to produce skeletal muscle myotubes from stem cells to create an in vitro disease model (Figure 3). In a direct application, pluripotent stem cell models of skeletal muscle have also been successfully used to develop a novel treatment for Duchenne muscular dystrophy (Moretti, 2020).


Conclusions


The promise and potential of stem technologies to advance biology, medicine and food production can only be fulfi lled if stem cell culture conditions are consistent, and accessible to research scientists and commercial operations alike. Exciting advances across multiple aspects of the stem cell workfl ow have streamlined processes to deliver products that are fully defi ned and animal-free. Furthermore, clinical translation of stem cell therapies and drug discovery are accelerated by the availability of GMP compliant reagents. The foundations are set for a bright future of discoveries and applications emerging from stem cell technologies.


The Authors


Dr William Hadlington-Booth is the business unit manager for stem cell technologies and the extracellular matrix at AMSBIO. Erik Miljan, PhD, is a pioneer in the development of cellular therapies for a range of degenerative and disease conditions. He holds a PhD in biochemistry from Hong Kong University. For further information please contact: William@amsbio.com


References


Moretti, A. F., et al. (2020). Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy. Nature Medicine, 26, 207–214.


Takahashi K., et al. (2006). Induction of pluripotent stem cells from mouse embryonic and adult fi broblast cultures by defi ned factors. . Cell, 126, 663-676.


Takayama, K. (2020). In Vitro and Animal Models for SARS-CoV-2 research. Trends in Pharmacological Sciences, 41. 513-517.


Sano, E., et al. (2021). Modeling SARS-CoV-2 infection and its individual differences with ACE2-expressing human iPS cells. Iscience, 24(5), 102428.


Promising progress is being made to create meat in the laboratory or what is commonly called ‘cultured meat’. Environmental concerns are driving the need for more sustainable meat production over traditional farming methods. Stem cell research in itself is reducing the need for the use of animals across multiple aspects as highlighted here. Producing cultured meat is straightforward in principle but faces many challenges in practice, for example maintaining the correct environment and stimuli for cultured cells to produce meat with the correct consistency and characteristics of the animal derived product. Stem cell cultures are expanded at scale in bioreactors and differentiated into skeletal muscle cells. These can be structured, using an edible scaffold for example, or used unstructured as the raw material to produce meat products (Figure 4). Tools and technologies are readily available to achieve this goal: expansion and differentiation of stem cells is highly effi cient. However, a key consideration is the cost of goods. Current technologies are too costly but these are pioneering times and research is moving at an exciting pace.


Figure 4. Meat manufactured in the laboratory is a viable alternative that can look and taste like meat harvested from farm animals.


Figure 2. Images of iPS cells in StemFit medium on iMatrix-511 A) before; and B) after cryopreservation infected with SARS-CoV-2 virus. Images courtesy of Kazuo Takayama, Kyoto University, Japan.


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