Paul J. Price, Ian Lyons and Shayne E. Boucher

1. Characterization • Stage-specific Ab • Flow Cytometry • Cytogenetics • Array Screening • RNAi • Isolation/Selection • Enzymatic digest • Nanobeads • Fractionation • FACS/Cell Sorter • Immunoselection • Media Technology • Classical Media • Specialty Media • Serum • Serum Replacer • Bioreactor • Cytokines • Expansion • Pluripotency • Differentiation • Maintenance • Cellular Signaling • Biopreservation • Vitrification • Germ Cell/BlastocystCryopreservation • Storage • Transport Solution Stem CellSystems • Cell Environment • Feeder Technologies • Attachment Factors • Cell-Cell Interaction • 3-D Matrices/Scaffolds • Models • Transgenesis • Expression Systems • Imprinting • Gene Reprogramming • Bioinformatics • Stem Cell Type • Species • Embryonic • Adult • Immortalized • Engineered Strategies for Development of Culture Media: Application to Embryonic and Adult Stem Cells Paul J. Price, Ian Lyons and Shayne E. Boucher GIBCO Cell Culture R&D, Invitrogen Co • 3175 Staley Road• Grand Island, New York 14072• USA Culture Media Design Strategy Embryonic Stem Cell Neural Stem Cell Mesenchymal Stem Cell Hematopoietic Stem Cell Culture Strategies Culture Strategies Culture Strategies Culture Strategies Basic strategy for designing stem cell culture media is to mimic metabolic conditions observed in vivo with some specialized modifications (see figure below). These modifications include adding substrates synthesized by organs, maintaining redox equilibrium, ameliorating lactate buildup, reducing glutamine-induced ammonia production, and maintaining region- or developmental stage-specific osmotic homeostasis. Special consideration needs to be given to handling, shelf-life, light exposure and storage temperature of media products. Finally, researchers need to select an appropriate experimental culture system that will allow them to study specific aspects of stem cell function. The interpretation of resulting findings will have to be weighed against the context of the experimental culture system utilized in the study. • Characterization • Stage-specific Ab • Flow Cytometry • Cytogenetics • Array Screening • RNAi • Characterization • Stage-specific Ab • Flow Cytometry • Cytogenetics • Array Screening • RNAi • Characterization • Stage-specific Ab • Flow Cytometry • Cytogenetics • Array Screening • RNAi • Isolation/Selection • Enzymatic digest • Nanobeads • Fractionation • FACS/Cell Sorter • Immunoselection • Media Technology • Classical Media • Specialty Media • Serum • Serum Replacer • Bioreactor • Isolation/Selection • Enzymatic digest • Nanobeads • Fractionation • FACS/Cell Sorter • Immunoselection • Media Technology • Classical Media • Specialty Media • Serum • Serum Replacer • Bioreactor • Isolation/Selection • Enzymatic digest • Nanobeads • Fractionation • FACS/Cell Sorter • Immunoselection • Media Technology • Classical Media • Specialty Media • Serum • Serum Replacer • Bioreactor - bFGF & EGF + T3 Red Blood Cells Oligodendrocyte Granulocytes Mesenchymal Stem Cells • Cytokines • Expansion • Pluripotency • Differentiation • Maintenance • Cellular Signaling • Cytokines • Expansion • Pluripotency • Differentiation • Maintenance • Cellular Signaling • Cytokines • Expansion • Pluripotency • Differentiation • Maintenance • Cellular Signaling + PDGF, RA or LIF • Biopreservation • Vitrification • Germ Cell/BlastocystCryopreservation • Storage • Transport Solution • Biopreservation • Vitrification • Germ Cell/BlastocystCryopreservation • Storage • Transport Solution • Biopreservation • Vitrification • Germ Cell/BlastocystCryopreservation • Storage • Transport Solution Macrophages Stem CellSystems Stem CellSystems Stem CellSystems Platelets HSC Neurosphere Neuron + 10% FBS, CNTF or SHH Dendritic Cells Precursor Cells Lymphocytes • Cell Environment • Feeder Technologies • Attachment Factors • Cell-Cell Interaction • 3-D Matrices/Scaffolds • Models • Transgenesis • Expression Systems • Imprinting • Gene Reprogramming • Bioinformatics • Cell Environment • Feeder Technologies • Attachment Factors • Cell-Cell Interaction • 3-D Matrices/Scaffolds • Models • Transgenesis • Expression Systems • Imprinting • Gene Reprogramming • Bioinformatics • Cell Environment • Feeder Technologies • Attachment Factors • Cell-Cell Interaction • 3-D Matrices/Scaffolds • Models • Transgenesis • Expression Systems • Imprinting • Gene Reprogramming • Bioinformatics Mature Cells • Stem Cell Type • Species • Embryonic • Adult • Immortalized • Engineered • Stem Cell Type • Species • Embryonic • Adult • Immortalized • Engineered • Stem Cell Type • Species • Embryonic • Adult • Immortalized • Engineered Astrocyte Bone Heart Tendon Adipose Stroma Neuron Modified from NIH Report on Stem Cell: Scientific Progress And Future Research Directions. Lactate NH3 Media Designs Media Designs Media Designs Media Designs Amino Acids Glycolysis Proteins PentosePO4 Pathway Carbo-hydrates Sugars Fatty Acids, Glycerol Fats intracellular compartment TCACycle Complex Nutrients extracellular compartment ATP, CO2, H20 Cellular Metabolism Past & Future Trends Past & Future Trends Past & Future Trends Past & Future Trends Anatomy of Culture Media In early neural stem cell (NSC) culture systems, researchers achieved and maintained NSC growth by using basal medium (i.e. D-MEM/F-12), simplified supplements (i.e. ITS and N-2), and growth factors (i.e. EGF and FGF-2)(Bottenstein and Sato, 1979; Espinosa-Jeffrey et al., 2002). Now, researchers are working towards a more robust and defined culture system that raises NSC yield, generates efficient clones, increases level of pluripotency, and expands lineage-restricted CNS precursors (Kallos et al., 2003). In the future, the focus will be on standardizing NSC culture methods. First, growth of NSC in 2-D monolayer cultures and 3-D neuro-spheres are being tested and validated to determine which system yields pluripotent stem cells that are more characteristic of NSC in vivo. Second, NSC may require customized culture conditions representative of brain regions or species that these stem cells are derived from. Third, researchers are investigating the plasticity of adult non-CNS lineage stem cells such as bone marrow-derived stem cells to generate bona fide NSC. Lastly, researchers are testing cytokine cocktails and inducible transgenes to yield specific CNS cell types for neurogenesis and transplantation studies. Mesenchymal stem cells (MSC), first discovered nearly three decades ago (Friedenstein et al., 1976), are viewed as a major class of cells now referred to as adult stem cells. Under traditional culture methodologies utilizing batches of fetal bovine serum with variable performance, MSC can grow and differentiate into a number of tissue phenotypes including bone, cartilage, adipose, tendon and, more recently, neurons and cardiomyocytes (Makino et al., 1999; Pittenger et al., 1999; Woodbury et al., 2000). Animal studies have shown that MSC can be expanded, differentiated and transplanted to correct tissue specific damage and genetic abnormalities (Pereira et al., 1995; Chen et al., 2001 ). A limited number of human clinical trials are underway to determine the safety and efficacy of using MSC and/or their ex vivo expanded progenitors in the treatment of human disorders. More defined and controlled culture systems including serum-free media and matrix technologies are needed to address regulatory concerns in utilizing MSC for cell therapy applications. Expansion and maturation of hematopoietic stem cells (HSC) to specific immune cell types is among the most mature of stem cell technologies. This was predicated on major long-term research and media development programs using enriched basal media formulations supplemented with sera. Presently, several serum-free formulations exist but these do contain animal-derived components such as serum albumin. Expansion and maturation signals for HSC are known and have been applied under serum-free conditions, but in many instances the response has been inefficient (Sandstrom et al., 1994). Future trends will focus on expanding and maturing HSC in an environment free of animal-derived components. Further knowledge of signaling pathways will improve responsiveness of HSC to external signals with increased specificity and efficacy (Maillard et al., 2000; Heng et al., 2004). Classical studies typically maintained mouse embryonic stem cells (mESC) on mouse fibroblast feeder layers in DMEM and screened batches of FBS (Robertson, 1992). The use of leukemia inhibitory factor (LIF) has been shown to replace the feeder layers (Williams et al., 1988) though most researchers still incorporate both LIF and feeders. Advances in mESC culture system has included the development of a serum replacement (i.e. KnockOut Serum Replacer, Goldsborough et al., 1998) that obviated the need for serum batch testing, and a modified D-MEM formulation developed to provide a lower osmotic environment preferred by mESC. These media specifications have been adopted for human ESC (hESC) and non-human primate ESC cultures, though clearly not optimized for these cell lines. In these human and primate systems, FGF-2 is required to maintain cells in an undifferentiated state (Amit et al., 2000). These cell lines appear more sensitive to variability in serum replacement lots. Several research initiatives are currently underway in optimizing and standardizing culture conditions as well as identifying and evaluating potential feeder-free systems for hESC lines. ChallengesCreate expansion & maturation culture systems containing no components of animal origin & efficient induction of signaling pathways. ChallengesDefine & test osmolarity, feeder conditions & media formulation to optimize culture conditions for understanding hESC biology & signaling pathways. ChallengesConstruct & standardize high quality NSC culture strategies to facilitate translation of research findings into cell therapy applications. ChallengesDevelop serum-free culture systems to expand, maintain & differentiate MSC into desired cell types for clinical applications. Acknowledgment: We thank Carol Berry, John Daley, Richard Fike, Thor Roalsvig, and Mary Lynn Tilkins for technical input and assistance in preparing this poster. 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