Genetic Engineering & Biotechnology News

MAY1 2015

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40 | MAY 1, 2015 | GENengnews.com | Genetic Engineering & Biotechnology News M esenchymal stem cells (MSCs) are self-renewing cells that differentiate into several ter- minally differentiated cell types. These cells have been isolated from multiple sources such as bone marrow, adipose tissue, peripheral blood, and other adult tissues. 1– 6 MSCs hold promise for treating many diseases and are being pursued in clinical trials. Emerging felds of interest for M S C s are cell therapy, regenerative medi- cine, and screening of candidate drugs. Therapeutic, Cost-Savings, and Other Benefts of MSCs In many cases, poor correlation between effcacy of candidate drugs in animal models and humans is observed. This leads to high attrition rates of candidate drugs from the developmental pipeline and contributes to large losses in revenue spent on animal model testing. The ability to isolate, expand, and differentiate human stem cells in vitro will expedite developmental timelines by al- lowing candidate drug testing on human cells at early stages, thereby better predicting how human populations may react to new and developing drugs. The ability to reproducibly isolate and expand these cell types should fa- cilitate the identifcation of candidate drugs earlier in the development process. Differen- tiating stem cells into various cell lines should also allow for more relevant toxicity testing. These achievements should ultimately lead to overall cost savings and decreased health risks in the future. In addition to drug product testing, several clinical trials have been initiated using stem cells in cell therapy treatments. Research has shown that the inherent properties of stem cells, such as differentiation potential, angio- genic potential, immunosuppression, or im- mune-privilege may be effective in the treat- ment of many diseases. Clinical trials using stem cells for the treatment of osteoarthritis, spinal cord injuries, Parkinson's disease, isch- emia due to stroke, cardiac arrests, or diabetes are seeing promising results. Obstacles Limiting the Use of MSCs For toxicology screening and cell ther- apy applications, large numbers of cells are needed. Expansion of adult stem cells is dif- fcult since they have a fnite life span and pluripotency can be lost. Due to the restricted number of population doublings, achieving maximal possible MSC expansion in the few- est passages is vital. Two-dimensional (2D) culture systems such as t-fasks, cell cubes/ factories, and roller bottles are common pro- duction platforms for vaccine and biologics manufacturing, as well as cell therapy. These systems are typically used for the expansion of cells to seed large bioreactors. Although well-established, these formats occupy a large footprint, are labor intensive, and susceptible to contamination problems due to numerous open handling steps. The Promise of Microcarrier-Expanded MSCs Microcarriers offer a large surface area for growth of anchorage-dependent cell types, and could thereby facilitate use of bioreactors for stem cell expansion in fewer passages. We conducted experiments to character- ize MSC expansion on fatware and fve Pall SoloHill microcarriers in stirred vessels. Re- tention of multipotency of the MSCs expand- ed in stirred culture was verifed by immu- nostaining with stem cell specifc antibodies and assessing their ability to differentiate into osteocytes and adipocytes. It was further illus- trated that MSCs can be expanded on various types of SoloHill microcarriers. The beneft of MSC expansion on mi- crocarriers is twofold. First, expansion on microcarriers allows growth on large surface areas within single containers, and second microcarrier expansion increases the ratio of apparent surface area to medium volume due to the fact that MSC growth on microcarriers outpaces growth on fatware. This is particularly important with stem cells grown in medium that contains expen- sive supplements. Therefore, the use of micro- carriers allows minimal passages for expan- sion of cells while decreasing the overall cost required to grow enough cells for a therapeu- tic dose in clinical trial treatments. We showed that multiple passages on microcarriers do not affect the ability of MSCs to differentiate into adipocytes and osteocytes. The ability to maintain pluripo- tency while expanding MSCs on microcar- riers for fve or six passages allows for the isolation of cells from bone marrow onto a T-150 fask. Cells expanded in this fashion can subsequently seed a small scale spinner culture, which could be used to seed a small bioreactor. For example, the maximal confuent cell density in a T-150 results in 2.5– 3 × 10 6 cells and 3.5– 4 × 10 4 cells/cm 2 on microcarriers. To seed a 200 mL spinner volume requires 3.1 × 10 6 cells using 5, 150 cm 2 /L. The maxi- mal densities on spinner cultures achieved here would result in enough cells for a mini- mum 10-fold expansion into a 2 L bioreac- tor, by the third passage after isolation and the second passage on microcarriers. Considering the recoverable cell numbers presented in this study, a 6.7 L bioreactor vol- ume (at 5, 150 cm 2 /L) would result in approx- imately 1 billion cells (~1 x 10 9 ). Assuming similar growth between small-scale spinners and bioreactors, a 2 L bioreactor could be used to seed a 20 L bioreactor, which would result in more than 10 billion cells from a sin- gle T-150 and multiple passages on microcar- riers. Additionally, increasing the microcarrier concentration beyond 5, 150 cm 2 /L would de- crease bioreactor volume required for a large number of recoverable MSCs. Work will continue to further characterize stem cells grown on SoloHill microcarriers and to include other stem cells. Additionally, work will continue to defne growth and ex- pansion conditions under animal component free environments, as well as direct expan- sion on microcarriers of isolated stem cells, thereby bypassing fatware tissue culture and increasing the number of available passages before senescence. Download the complete Application Note, providing details of this study, at: www.pall.com/pdfs/Biopharmaceuticals/ Microcarriers_Mesenchymal_StemCell_ Expan_USD2976_AN.pdf n References 1. Friedenstein AJ, Gorskaja JF, Kulagina NN. Fibro- blast precursors in normal and irradiated mouse he- matopoietic organs. Exp Hematol 1976, 4: 267-274. 2. Fraser et al. Fat tissue: an underappreciated source of stem cells for biotechnology. Trends Biotechnol 2006, 24: 150-154. 3. Cao C, Dong Y. Study on culture and in vitro os- teogenesis of blood-derived human mesenchymal stem cells. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2005. 19: 642-647. 4. Griffths Mu, Bonnet D, Janes SM. Stem Cells of the Aveolar epithelium. Lancet. 2005, 366: 249-260. 5. Beltrami et al. Adult cardiac stem cells are multi- potent and support myocardial regeneration. Cell. 2003, 114: 763-776. 6. Pittenger et al. Multilineage potential of adult hu- man mesenchymal stem cells. Science. 1999, 284: 143-147. © 2015, Pall Corporation. Pall, the Pall logo, and SoloHill are trademarks of Pall Corporation. ® indicates a trademark registered in the USA and TM indicates a common law trademark. Pall Life Sciences Mark Szczypka Director of Applications and New Products mark_szczypka@pall.com www.pall.com APPLICATION NOTE Advertorial Expansion and Characterization of Mesenchymal Stem Cells on Pall SoloHill ® Microcarriers Mark Szczypka, David Splan, and Heather Woolls

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