1. Source: http://www.scq.ubc.ca/stem-cell-bioengineering/ CULTURE SYSTEMS FOR IN VITRO EXPANSION AND DIFFERENTIATION OF STEM CELLS Biomedical Engineering Applications Culture Media Small scale culture systems Large scale culture system It has not yet been developed a large scale culture system completely efficient: it is still difficult to assure the physiological conditions needed for cell growth and to achieve an efficient homogeneity within the bioreactor without damaging the cells. The success of scale-up is dependent on immobilization techniques. Microcarriers These supports are typically spherical, made of gelatine, collagen or other material either natural or synthetic. They are used to increase the surface area available for cell growth. Cells grown in the interior of their pores are protected from fluid shear force. Microencapsulation Cells can be encapsulated in biocompatible polymeric matrices, where they are protected from the mechanical force generated by fluid flow, thus the system can be agitated in a higher speed, improving the mass transfer within the system. Bioreactors Stem cells There are three criteria used to identify a stem cell (SC): ability of self-renewal, multilineage differentiation capacity and in vivo engraftment potential. These characteristics distinguish SC from the almost 200 cell types in an adult mammal. SC can be divided according to their potency and origin. Origin Embryonic SC Fetal SCAdult SC Static The most used for expansion of SC e.g. T-Flask, Petri dishes Dynamic e.g. Spinner Flask Many limitations: Lack of homogeneity; hard to monitor and control; limited production. Perfusion chamber bioreactor Culture medium continuously renewed Cells retained in the bioreactor Small culture chambers with a layer of stromal cells Successful expansion of Hematopoietic SC Stirring Homogeneous environment Overcome problems of static culture systems Keep the cells in suspension Effect on cellular membrane / microenvironment Fixed-bed 3-D scaffolding for cell attachment Co-culture with Hematopoietic SC (immobilised in microcarriers) Fluidised-bed Improvement of the performance of the fixed-bed system Better homogeneity Easier scale-up SC applications in Tissue Engineering and Regenerative Medicine Use of the extracellular matrix of decellularized organs as a scaffold Alternative bioreactor configurations Assuring nutrient supply and avoiding the accumulation of metabolic by-products Glucose and glutamine as nutrients, lactate and ammonia as by-products. pH of the culture (7.0-7.5) small variations in pH can substantially affect the performance of SC cultures. Dissolved oxygen In a compromise, to avoid toxicity and fulfill oxygen needs. The use of animal serum Cellular grow and adhesion. Supportive feeder cells The cultivation of stem cells has three main objectives: (i) sustaining the self regeneration properties, (ii) maintaining the capability of differentiation (iii) and enabling cryopreservation for maintaining the established cell lines. Isolation Physical Properties CentrifugationSize selection Biological Properties Magnetic Cell Sorting (MACS) Fluorescent- Activated Cell Sorting (FACS) Culture Control of Culture Media Bioreactors 3D scaffolds/matrices Enhance correct differentiation of SC Mimic physiological environment Entrapment of extra-cellular matrix Structural support Addition of biomolecules Imaging In vivo Magnectic Ressonance Imaging (MRI; microMRI) Positron Emission Tomography (PET; microPET) In vitro Microscopy Fluorescente Microscopy Cell Processing ImagingBiomaterials & Tissue Engineering Basic research; Pharmaceutical research; Cellular therapy; Gene Therapy; Tissue engineering. Due to their proliferative capacity and ability to become specialized into almost any cell of an organism, stem cells hold the promise of being able to repair or replace damaged or destroyed cells and tissues. a) Rat heart matrix after process of decellularization; b) Recellularization of the same heart using rat stem cells. Acknowledgements: Dr. Cláudia Lobato da Silva Departamento de Engenharia Química e Biológica – DEQB/IST Totipotent The fertilized egg and cells resulting from its division, within 3-4 days Pluripotent Cells from the inner mass of the embryo with the ability to give rise to cell of all germ layers Multipotent Cells of a certain tissue with origin in one specific germ layer. Stirred-tank bioreactor Amorim, Inês 1 ; Bandeira, Adriana 2 ; Pedrosa, Catarina 3 1 65099, ines.amorim@ist.utl.pt; 2 66283, adriana.bandeira@ist.utl.pt; 3 65091, catarinapedrosa@ist.utl.pt Quantum dot labeled human mesenchymal stem cells undergoing proliferation. Source: Kherlopian, Armen R, et al. “A Review of Imaging Techniques for Systems Biology.” 2008 Bioluminescense and PET images of mouse injected with ESC and consequent teratoma formation. Source: “The journal of nuclear medicine”, Vol. 48, No. 12, December 2007. Spurce:http://www.nature.com/embor /journal/v6/n4/full/7400383.html. Spurce: Bioengineering Research Group, IBB Stirred suspension bioreactor Spurce: Bioengineering Research Group, IBB Source:http://probes.invitrogen.com/resources /education/tutorials/4Intro_Flow/player.html. Fixed/fluidised-bed bioreactor Source: Lobato, Cláudia da Silva. “Stem Cells for Cell and Tissue Engineering., Cell and Tissue Engineering Classes, IST, 2007-2008 Perfusion chamber bioreactor Source: Lobato, Cláudia da Silva. “Stem Cells for Cell and Tissue Engineering., Cell and Tissue Engineering Classes, IST, 2007-2008 Conclusion The future of stem cell engineering and all the promising applications of these cells rely on the capacity of scientist to successfully isolate, expand and differentiate them. Many challenges must be overcome in a wide variety of areas that include: i) understanding the mechanisms of differentiation of stem cells or how to maintain them undifferentiated; ii) development of efficient protocols either for isolation, expansion and differentiation of stem cells; iii) development of culture systems; iv) and applications of the existent protocols and culture systems at clinical scale. Mestrado Integrado em Engenharia Biomédica Introdução à Engenharia Biomédica 1º ano, 1º semestre 2008/2009 References: Cabral, M. S. Joaquim, Sommer Bruno Ferreira, e Cláudia Lobato da Silva. “Reactores para Cultura de Células Estaminais.” Cap. 18 em Reactores Biológicos: Fundamentos e Aplicações, de Manuela M. Fonseca e A. José Teixeira. Lidel. Kherlopian, Armen R, et al. “A Review of Imaging Techniques for Systems Biology.” 2008. Lobato, Cláudia da Silva. “Stem Cells for Cell and Tissue Engineering., Cell and Tissue Engineering Classes, IST, 2007-2008