This document discusses primary cells and their use in biomedical research. Primary cells are isolated directly from tissues and have important advantages over cell lines, including greater biological relevance. Primary cells are used in applications such as biomarker research, drug development, immunotherapeutics, and tissue engineering. The document reviews techniques for working with primary cells, including isolation, culture, differentiation, and characterization using methods like flow cytometry and immunoassays. Primary cells provide a more physiologically relevant model for research compared to cell lines.

1. 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

3. ConfidentialYour 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

4. ConfidentialYour Reliable Source of Human Primary Hematopoietic Cells and Services
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 type
Chromosomal aberrations
Identity is questionable
Heterogeneous cell population (PB-MNC)
Variability
Can select specific donor characteristics
Can 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 fresh
High viability
No specialized media
Limited lifespan

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Primary cells vs. Cell lines
Applications
Cell Biology
Disease mechanisms
Drug development
Biomarkers
Tissue 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-arrays
1. Fabricate DNA micro-spots
2. Cancer cell death after 16 hr α-CD20 treatments
3. Drug dosage data
100 um
Blue-Live cancer cells
Purple-Dead cancer cells
Red- 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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Immunotherapeutics: Autoimmune, HIV
Hyaluronic acid implicated in inflammation
Green- Mesenchymal stem cell expressing hyaluronic acid
Red- 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 Phenotype100 um
CD68 CD80
CD163CD68
Inactive Active
M1 Phenotype
Lineage Tree

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Tissue Culture, Differentiation, Characterization
Multipotency 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
TargetA-APC
Target B-PE
TargetC-FITC
TargetE-PacificBlue
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 ekelly@allcells.com or
sales@allcells.com
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