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Your Reliable Source of Human Primary CellsPrimary Cells 101
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Webinar Outline
•What are primary cells?•Primary Cells vs Cell Lines•Examples of primary cell applications•How to work with primary cells•Q&A
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Your Reliable Source of Human Primary Hematopoietic Cells and Services
The diverse history of Cell Culture
1885: Chicken embryology
1940s: Vaccines
1960s: Embryonic stem cells
1970s: Recombinant DNA
1951 HeLa
Cell Line
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What are Primary Cells?
Subculture/ passage
transformationtumor
• Cell Lines• Continuous Cell lines
• Primary Cells• Primary Hematopoietic Cells
Lymphocytes
HeLa Cells
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Primary cells vs. Cell lines
Biological relevanceBiological relevance
Uniform cell typeChromosomal aberrationsIdentity is questionable
Heterogeneous cell population (PB-MNC)
VariabilityCan select specific donor characteristicsCan order many vials from a single donor
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Primary cells vs. Cell lines
Access and Ease of useAccess and Ease of use
Low cost (buy once) Easy to keep alive
Frozen or freshHigh viabilityNo specialized media
Limited lifespan
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Primary cells vs. Cell lines
Applications
Cell BiologyDisease mechanismsDrug developmentBiomarkersTissue engineering
Reagent development
Manufacturing/ Industrial
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Examples of Primary Cell Applications
1. Biomarkers•$53.6 bn (2018) •Cancer biomarkers-up 18.26% by 2019
2. Drug screening•$16.6 bn (2015)
3. Immuno-therapeutics• $67.9 bn (2018)
4. Tissue Engineering•$10.6 bn (2018)•Americas hold largest market share
Source: MarketWatch (Dow Jones)
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Biomarker Research: Drug Resistance
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Biomarker Research: Drug Resistance
Gandhi, et al. (2014) British Journal of Haematology. 164: 233-244.
• Immunomodulatory drugs (IMiDs) are used to treat hematological pathologies
• Cereblon mRNA levels correlate with IMiD resistance
Wild Type IMiD Resistant
Microscopy of Cell lines
Cereblon levels vs. Drug resistance
Wild Type IMiD Resistant
100 um
CD138+ Multiple Myeloma Bone MarrowCD138+ Multiple Myeloma Bone Marrow
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Drug Screening: Cytotoxicity Micro-arrays• Test drug compounds for effects on cells• Detect cell death, phenotype changes, specificity, protein expression
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Drug Screening: Cytotoxicity Micro-arrays1. Fabricate DNA micro-spots
2. Cancer cell death after 16 hr α-CD20 treatments
3. Drug dosage data
100 um
Blue-Live cancer cellsPurple-Dead cancer cellsRed- Dead PBMNC
Hsiao SC, Liu H, Holstlaw TA, Liu C, Francis CY, et al. (2013) PloS ONE 8(6) e66739.
Peripheral Blood Mononuclear CellsPeripheral Blood Mononuclear Cells
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Immunotherapeutics: Autoimmune, HIV
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Your Reliable Source of Human Primary Hematopoietic Cells and Services
Immunotherapeutics: Autoimmune, HIV
Hyaluronic acid implicated in inflammation
Green- Mesenchymal stem cell expressing hyaluronic acidRed- Leukocytes
Engineered B-Cells produce anti-HIV antibodies
Kota, D.J., et al. (2014) Scientific Reports (4): 4565.
100 um
25 um
Luo, X.M., et al. (2009) Blood (113): 1422-1431.
Bone Marrow, Cord Blood CD34+ cellsBone Marrow, Cord Blood CD34+ cells
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Tissue Engineering: Vascularized Grafts
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Tissue Engineering: Vascularized Grafts• A novel methodology for co-implantation of CD34+ hematopoietic progenitors
and mesenchymal stem cells (MSC)
Moioli, E. K. et al. (2008) PLoS One. 3 (12); e3922.
Bone Marrow, Bone Marrow CD34+Bone Marrow, Bone Marrow CD34+
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How to work with Primary Cells
• Cell Isolation and Handling Procedures• Tissue culture, differentiation and characterization• Downstream processing
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Purified Cell Isolation and Handling
Instructional videos on cell handling available at AllCells.com
Immunomagnetic cell separation Whole Tissue Isolation
Freezing Primary CellsThawing Primary Cells
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Tissue Culture, Differentiation, Characterization
100 um 100 um
T-cell activation
Macrophage polarization
M2 Phenotype 100 um
CD68 CD80
CD163CD68
Inactive Active
M1 Phenotype
Lineage Tree
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Tissue Culture, Differentiation, CharacterizationMultipotency of Bone Marrow Mesenchymal Stem Cells
100 um
Microscopy images of differentiation
Bench-top bioreactors for MSC tissue engineering
Perfusion co-cultures
Mechanical-based cues during culture
Lineage Tree
Cardiomyocytes
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Tissue Culture, Differentiation, Characterization
Targ
et A
-APC
Target B-PE
Targ
et C
-FIT
C
Targ
et E
-Pac
ific
Blue
Target F-APC-CY7
Propidium Iodide (PerCP)
SSC-
A
Multi-color Flow Cytometry and Fluorescence-Activated Cell Sorting (FACS)
Data readout:
BD FACS Canto II
Target D-PE-CY7
BD FACS Aria III
Relative equipment:
Cell Sorting
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Tissue Culture, Differentiation, Characterization
Bio Rad Bio-plex 200
Meningococcal Vaccine Research
Multiplex Luminex Cytokine analysis
Relevant Equipment:
Example: Applications:
Mechanism:
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Conclusions
• Primary cells lines provide a more relevant system for biomedical research in contrast to cell lines
• Primary cells are sourced via immunomagnetic separation and whole tissue isolation
• Primary cells are used in multifaceted research involving biomarkers, drug development, immunology and tissue engineering
• After isolation, primary cells can be cultured, differentiated and characterized using flow cytometry, immunoassays, and many other techniques
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Erin Kelly, PhD.
Bioservices Supervisor
Please direct offline questions to [email protected] or [email protected]
www.allcells.com/technical-support/
www.allcells.com/support/webinars/
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