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

Invitrogen Corporation • 1600 Faraday Avenue • Carlsbad, California 92008 USA • Telephone: 760 603 7200 • FAX: 760 602 6500 • Toll Free Telephone: 800 955 6288 • E-mail: [email protected] • www.invitrogen.com

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

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.

Basal Media Supplements

DMEM Fetal Bovine Serum

KnockOut™ D-MEM ES Qualified FCS

-KnockOut™ Serum

Replacement

Strategies for Development of Culture Media: Application to Embryonic and Adult Stem Cells

Past & Future Trends

Media Designs

Culture Strategies

Embryonic Stem Cell

ChallengesDefine & test osmolarity, feeder conditions & media formulation to optimize culture conditions for understanding hESC biology & signaling pathways.

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.


DMEM/F12 N2

Neurobasal™ B27

Neurobasal™ -A B27 w/o RA


Media Designs

Culture Strategies

Neural Stem Cell

ChallengesConstruct & standardize high quality NSC culture strategies to facilitate translation of research findings into cell therapy applications.


Macrophage-SFMSignaling molecules for guidance and function

AIM-V®Signaling molecules for guidance and function

StemPro®-34 SFMSignaling molecules for guidance and function


MEM BSA, Insulin, Transferrin, LDLs, PDGF, EGF

DMEM FBS

McCoy’s F5A FBS

Stem CellSystems

Isolation/Selection•Enzymatic digest

•Nanobeads•Fractionation

•FACS/Cell Sorter•Immunoselection

Media Technology•Classical Media•Specialty Media

•Serum•Serum Replacer

•Bioreactor

Biopreservation•Vitrification

•Germ Cell/BlastocystCryopreservation

•Storage•Transport Solution

Cell Environment•Feeder Technologies•Attachment Factors•Cell-Cell Interaction

•3-D Matrices/Scaffolds

Cytokines•Expansion

•Pluripotency•Differentiation•Maintenance

•Cellular Signaling

Models•Transgenesis

•Expression Systems•Imprinting

•Gene Reprogramming•Bioinformatics

Stem Cell Type•Species

•Embryonic•Adult

•Immortalized•Engineered

Characterization•Stage-specific Ab•Flow Cytometry

•Cytogenetics•Array Screening

•RNAi

extrace

llular com

partm

ent

intracellu

lar co

mp

artmen

t

ATP, CO2, H20

Complex Nutrients

Proteins

Carbo-hydrates

Fats

Amino Acids

Sugars

Fatty Acids,

Glycerol

Glycolysis

Lactate

PentosePO4

Pathway

NH3

TCACycle

Cellular MetabolismCellular Metabolism

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.

Anatomy of Culture Media

Components Issues

WaterSource; Purification; Seasonal Variation

Balanced Salt SolutionOsmotic & Ionic Equilibrium;

Metal Ions; Buffer

Energy SourcesSugars; Biosynthesis;

Lactate Buildup

GlutamineATP Production; NH3

Production; Biosynthesis

Amino AcidsBiosynthesis; Chelator; Buffer; Energy Source

VitaminsMetabolic Functions; Lipid

Synthesis; e- Transport

SerumCell Attachment; Growth; Buffer; Toxin Inactivation

Advanced AdditivesSerum Replacement; Lipids;

Attachment Proteins

Neurosphere

Oligodendrocyte

- bFGF & EGF + T3

Neuron

+ PDGF, RA or LIF

Astrocyte

+ 10% FBS, CNTF or SHH


ChallengesDevelop serum-free culture systems to expand, maintain & differentiate MSC into desired cell types for clinical applications.

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.


ChallengesCreate expansion & maturation culture systems containing no components of animal origin & efficient induction of signaling pathways.

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).

Acknowledgment: We thank Carol Berry, John Daley, Richard Fike, Thor Roalsvig, and Mary Lynn Tilkins for technical input and assistance in preparing this poster.

Media Designs

Culture Strategies

Mesenchymal Stem Cell

Stem CellSystems






•Bioreactor

















•RNAi

Media Designs

Culture Strategies

Hematopoietic Stem Cell

Precursor Cells

Mature Cells

Red Blood Cells

HSC

Macrophages

Platelets

Granulocytes

Dendritic Cells

Lymphocytes

Stem CellSystems






•Bioreactor

















•RNAi

Stem CellSystems






•Bioreactor

















•RNAi

Modified from NIH Report on Stem Cell: Scientific Progress And Future Research Directions. Bone Heart Tendon Adipose Stroma Neuron

Mesenchymal Stem Cells

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

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