SFN 2003

1
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 RD, 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.
Invitrogen Corporation 1600 Faraday Avenue
Carlsbad, California 92008 USA Telephone 760
603 7200 FAX [phone] Toll Free
Telephone [phone] E-mail
tech_service_at_invitrogen.com www.invitrogen.com