May 1, 2015 (Vol. 35, No. 9)
Mesenchymal stem cells (MSCs) are self-renewing cells that differentiate into several terminally 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 fields of interest for MSCs are cell therapy, regenerative medicine, and screening of candidate drugs.
Therapeutic, Cost-Savings, and Other Benefits of MSCs
In many cases, poor correlation between efficacy 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 allowing 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 facilitate the identification of candidate drugs earlier in the development process. Differentiating 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, angiogenic potential, immunosuppression, or immune-privilege may be effective in the treatment of many diseases. Clinical trials using stem cells for the treatment of osteoarthritis, spinal cord injuries, Parkinson’s disease, ischemia due to stroke, cardiac arrests, or diabetes are seeing promising results.
Obstacles Limiting the Use of MSCs
For toxicology screening and cell therapy applications, large numbers of cells are needed. Expansion of adult stem cells is difficult since they have a finite life span and pluripotency can be lost. Due to the restricted number of population doublings, achieving maximal possible MSC expansion in the fewest passages is vital. Two-dimensional (2D) culture systems such as t-flasks, cell cubes/factories, and roller bottles are common production 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 characterize MSC expansion on flatware and five Pall SoloHill microcarriers in stirred vessels. Retention of multipotency of the MSCs expanded in stirred culture was verified by immunostaining with stem cell specific antibodies and assessing their ability to differentiate into osteocytes and adipocytes. It was further illustrated that MSCs can be expanded on various types of SoloHill microcarriers.
The benefit of MSC expansion on microcarriers 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 flatware.
This is particularly important with stem cells grown in medium that contains expensive supplements. Therefore, the use of microcarriers allows minimal passages for expansion of cells while decreasing the overall cost required to grow enough cells for a therapeutic 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 pluripotency while expanding MSCs on microcarriers for five or six passages allows for the isolation of cells from bone marrow onto a T-150 flask. 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 confluent cell density in a T-150 results in 2.5–3 × 106 cells and 3.5–4 × 104 cells/cm2 on microcarriers. To seed a 200 mL spinner volume requires 3.1 × 106 cells using 5,150 cm2/L. The maximal densities on spinner cultures achieved here would result in enough cells for a minimum 10-fold expansion into a 2 L bioreactor, 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 volume (at 5,150 cm2/L) would result in approximately 1 billion cells (~1 x 109). 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 single T-150 and multiple passages on microcarriers. Additionally, increasing the microcarrier concentration beyond 5,150 cm2/L would decrease 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 define growth and expansion conditions under animal component free environments, as well as direct expansion on microcarriers of isolated stem cells, thereby bypassing flatware tissue culture and increasing the number of available passages before senescence.
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Pall Life Sciences
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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 osteogenesis of blood-derived human mesenchymal stem cells. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2005. 19: 642-647.
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5. Beltrami et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. Cell. 2003, 114: 763-776.
6. Pittenger et al. Multilineage potential of adult human mesenchymal stem cells. Science. 1999, 284: 143-147.
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