Vital role of 3-D scaffolding and Oxygen transport on Human Mesenchymal stem cells subjected to perfusion Bioreactor

1. Vital role of 3-D scaffolding and Oxygen transport on Human Mesenchymal stem cells subjected to perfusion Bioreactor Contents Personal Page Sub-topics and Papers cited Executive summary Background information 3-D scaffolding Oxygen transport Comparison of papers Personal comments By Zane Greaves (3101 4945)

2. Personal Page My Name is Zane Greaves. I am Majoring in Molecular biology and biotechnology. I decided to act on this particular topic as I have a profound interest in stem cells. Keeping to the guidelines of this task, I needed to refine my topic more to microbial processes. The closest, while still being about stem cells, was tissue engineering in a bioreactor.

3. Topics and Papers cited • Feng Zhao, Teng Ma., Perfusion Bioreactor System for Human Mesenchymal Stem Cell Tissue Engineering: Dynamic Cell Seeding and Construct Development, Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, Florida; 13 May 2005 in Wiley InterScience DOI: 10.1002/bit.20532 • Feng Zhao, PragyansriPathi, Warren Grayson, Qi Xing, Bruce R. Locke, and Teng Ma, Effects of Oxygen Transport on 3-D Human Mesenchymal Stem Cell Metabolic Activity in Perfusion and Static Cultures: Experiments and Mathematical Model., Department of Chemical and Biomedical Engineering, FAMU-FSU College of Engineering, Florida State University, Tallahassee, Florida 32310., Biotechnol. Prog. 2005, 21, 1269-1280 Perfusion Bioreactor System for Human Mesenchymal Stem Cell and 3-D scaffolding Perfusion Bioreactor System for Human Mesenchymal Stem Cell and oxygen transport

4. Executive summary The perfusion bioreactor is one such bioreactor for cell growth. It is has the potential of multiple tissue engineered construction, and homogeneous tissue development. It is relatively simple to operate and has the potential in clinical use. Tissue constructs regenerated from donors’ cells (stem cells) can be used to replace damaged or defective tissues and have broad clinical applications for a wide range of diseases. To establish such tissue constructs, 3-D scaffolding, and sufficient oxygen supply and transport into the cells are crucial. The following provides an understanding of the such at a simple level of understanding.

5. Background A Bioreactor is a device or system that is engineered that can contain and support an optimal active biological environment. These devices can be used to grow mammalian cells; specifically Stem cells. Bioreactor systems can be used to provide suitable environments for supporting functional engineered tissue constructs from stem cells. Stem cells are undifferentiated biological cells (self-renewal) that can be differentiated into specialised cells (Potency). Human mesenchymal stem cells (hMSCs) have the unique potential to develop into functional tissue constructs as a substitute to a vast selections of tissues of humans that have been damaged through such things as disease or injury. A blastocyst is an early stage embryo, four to five days old in humans and consisting of around 50-150 cells. The other type of stem cells are adult stem cells which are found in various tissues

6. Background Embryonic stem cells give rise to all cell derivatives of three primary germ layers: ectoderm, endoderm, and mesoderm. The perfusion bioreactor is one such bioreactor for cell growth. It is has the potential of multiple tissue engineered construction, and homogeneous tissue development. It is relatively simple to operate and has the potential in clinical use. They have been proven to enhance cell survival, growth, and function. High proliferation potential and plasticity make hMSCsappealing cell supply for tissue engineering. “Tissue constructs regenerated from donors’ cells can be used to replace damaged or defective tissues and have broad clinical applications for a wide range of diseases. Recent advances in human stem cell research demonstrate that human stem cells have unique properties that are important for the development of engineered tissue constructs, including high proliferation rates and self-renewal capacity, maintenance of the unspecialized state, and specialized differentiation under given conditions. Mesenchymalstem cells (MSCs) isolated from adult bone marrow possess a high capacity for self-replication and have the potential to differentiate into osteogenic, chondrogenic, adipogenic, and tenogeniclineages when placed in an appropriate environment” (Feng Zhao et al., 2005)

7. 3D-scaffolding in Perfusion Bioreactor System for Human Mesenchymal Stem Cell summary The results of article one show that differentiation potential of hMSCs are maintained after continuous growth. This allows for a construct to support such growth in a three dimensional environment. This is where the 3-D scaffolding construct is effective. The bioreactor system’s 3-D scaffolding not only provides efficient nutrient supply and metabolite removal (flow through for cells), it also delivers a physiochemicaland biochemical environment inductive to tissue growth. exploitation of 3-D scaffolds is a essential piece of tissue engineering tactic, and plays an indispensable function for viable and efficient tissue fabrication.

8. Oxygen transport in Perfusion Bioreactor System for Human Mesenchymal Stem Cell summary Aerobic respiration is a necessity for the MHSC growth and development. It is therefore important that these cells receive enough oxygen for sufficient growth and development. identical tissue development requires well-timed deliverance of nutrients and oxygen, while also removing metabolites. “Oxygen transport through the bulk liquid layer by convection and diffusion.”(Feng Zhao, et al. 2005) to the cells with a percentage of carbon dioxide and oxygen similar to that of the human blood dissolved gas percentage. “The specific oxygen consumption rate for the constructs in the perfusion bioreactor was found to decrease from 0.012 to 0.0017 ímol/106 cells/h as cell density increases, suggesting intrinsic physiological change at high cell density.” (Feng Zhao, et al. 2005) this paves way for a construct that would allow for the delivery of oxygen to the cells; 3-D scaffolding.

9. Comparison results from this study provide insight into the role of oxygen transport in hMSC proliferation and metabolism in 3-D constructs, which is important in optimizing culture conditions for hMSC tissue engineering. “Sustaining a spatially uniform construct development at high cell density is an important aspect of hMSC tissue engineering. Results from this study show that culture environment has significant influences on cell growth and metabolisms. The 3-D constructs grown in static cultures developed significant oxygen gradients and have the tendency to give rise to spatially non-uniform and inconsistent construct development, highlighting the need to optimize the culture environment for functional hMSC construct development.”2

10. Personal Comments hMSCs are an attractive cell source for tissue engineering purpose because they can be attained from healthy adults, have high proliferation capability, and multi-lineage differentiation potential (potency). The challenge, however, is to develop hMSCs from a restricted source to adequate measures for therapeutic appliance. This would imply that a larger scale of the bioreactor system would be needed. Up-scaling holds potential issues in cost, space, and critical levels of optimal biochemical and physicochemical factors in accordance to the increased size. Once, however, the potential issues are sorted out, this system will provide an indispensible tool in tissue engineering and could lead to other uses in the medical industry.

11. Additional information • There are a number of physicochemical and Biochemical implementaion that need to be address for stem cell bioreactor success: • Physicochemical • pH values above or below the physiological levels of the cells may be deterimental to cell growth • Temperature is typically mimiced at a human body temperature of 37 degrees celcius • Osmotic pressure has a major affect on the cell viability and cell numbers • Hydrodynamic shear stress occurs in stirred bioreactors. This due to the cells, in absence of gas bubbles, having the same size as the eddy created against the cell surface/area. Shear stress will be greater with increasing impeller diameter, rpm, and impeller location and geometery, and probes. Papoutsakis ET: Fluid-mechanical damage of animal cells in • bioreactors. Trends Biotechnol 1991, 9:427-437. • Oxygen tension has shown to have an effect on the rate of differentiation and proliferation. Human MSCs have been shown to grow at a higher rate under 2% O2 than at 20% O2, exibiting greater retension of MSC markers and formation of CFU-F (see Forsyth NR, Musio A, Vezzoni P, Simpson A, Noble BS, McWhir J: Physiologic oxygen enhances human embryonic stem cell clonal recovery and reduces chromosomal abnormalities. Cloning Stem Cells 2006, 8:16-23. ) lower O2 decreased chromosomal damages (and potentioanly abnormalities) as well. • Perfusion or frequent feeding enhances culture performance by replacing depleted nutrients and/or removing inhibitory metabolic byproducts. These two factors along with gas-permeable culture surfaces can increase the expansion of MSCs, ESCs, and mammalian epithelial stem cells while maintaining stem cell potential Zhao F, Ma T: Perfusion bioreactor system for human mesenchymal stem cell tissue engineering: dynamic cell seeding and construct development. BiotechnolBioeng 2005, 91:482-493.

12. Additional information • Biochemcial • Nutrients are important n cell metabolism and so must be supplied and monitored. Glucose and glutamine are considered the main sources fro cell energy, being utilised for carbon and nitrogen for cell functions, metabolism and biosythesis • Metabolic waste products, especially lactate and ammonia, may inhibit cell growth and should be tightly controlled.Fernandes TG, Fernandes-Platzgummer AM, da Silva CL, Diogo MM, Cabral JMS. Kinetic and metabolic analysis of mouse embryonic stem cell expansion under serum-free conditions. BiotechnolLett 2010b;32(1):171–9. • Growth factors and Cytokines are signaling proteins that modulate a wide range of cell functions, including self-renewal, differentiation or survival. For example, LIF and BMP4 can be used in combination to sustain mouse embryonic stem cells in culture, while a cocktail of growth factors composed of SCF, Flt-3 L and TPO can be used for the ex-vivo expansion of HSCs.van der Sanden B, Dhobb M, Berger F, Wion D. Optimizing stem cell culture. J Cell Biochem2010;111(4):801–7.

13. Additional information The following table shows the different types of batch bioreactors

14. Additional information • Stem cells are defined in two parts; the ability to differentiate, and their potency. The stem cells unique ability is that of a precursor cell, giving rise to other cell types. Its ability to give rise to x amount of cells is its measurement of potency. • Having viable cell lines is critical for the concept of using embryonic stem cells from bioreactors. Issues faced are in the genetic abnormalities that occur; specifically aneuploidy, which is defined as an abnormal number of chromosomes. An unusual number of chromosomes would have an unknown or undesirable differentiation development of the stem cell. • A serum free medium with BFGF was seen to increase HSC potency and differential state when compared to serum containing medium (KnockOut Dulbecco’s modified Eagle’s medium with additives) and BFGF (Human recombinant basic fibroblast growth factor). • Enhancing the defined characteristics of stem cells for therapeutic cloning and reproductive cloning would, if used correctly and remain un-abused, assist in human health studies and medical practises. Therapeutic cloning looks at using stem cells to observe human development and from this the potential to treat diseases. Whereas, more controversially, reproductive cloning can be seen as cloning human beings. The latter could be useful as a means of cultivating and harvesting organs for individual surgeries. Laws and ethical views vary between countries and their states.