Isolating Primary Cells 101

allcells
Isolating Primary Cells 101: Which viability
method is best for your research?
Ask the Experts Series #1
AllCells - Nexcelom Bioscience
Exclusive Webinar Event
2/13/2014
James Lee and Leo Chan, Ph.D.
Company Information

• Provider of image
cytometry technology
for cell concentration
and viability
• Leo Chan, Ph.D.

• Provider of primary
cells & bioservices for
pre-clinical research
• James Lee
Important Questions to Address
• What is the process
used for obtaining cells
from bone marrow?
• What is the process
used for obtaining cells
from peripheral blood?

• What is the best and
most accurate cell
counting and viability
method for primary
cells?
• How do we select the
appropriate method for
viability measurement?
Key Points to Take Away
• AllCells key takeaways: decrease variability, increase speed and
accuracy
• Nexcelom Bioscience key takeaways: the best method for
concentration and viability measurement using different
Cellometers, which is cell sample dependent
Company Information
ALLCELLS is a leading B2B provider of hematology-based cells & services
to the world’s life science research community for the past 16 years.



Products: >800 healthy & diseased human tissue and primary cell
products that enable drug discovery, stem cell research, and
advancement of therapies



Bioservices: Applications of hematology /
immunology expertise; cell-based assay
services and customized research studies



Core Competencies: Hematology, immunology, human tissue
procurement (healthy & diseased), large-scale cell isolations &
characterization, stem cell expansion & differentiation



Operations: Alameda, CA (HQ), IRB approved collection
programs, Tissue Bank and Biologics Licenses
Primary Cells - Uses
•
•
•
•
•

Drug Development – Pre-clinical Development
Predictive Toxicology
Stem Cell Research/Development
Regenerative Medicine
Medical Device Validation
Primary Human Cells by AllCells
• Normal Peripheral Blood -> MNC -> T Cells
(untouched)
• Bone Marrow -> MNC -> CD34+
(organ>isolation steps-> cell types)
(Cell viability, counting methods suitable for
each step)
Equipment Utilized
Miltenyi’s Separation Units

CliniMACS Prodigy
AutoMACS Pro
Separator
CliniMACS Plus
Pluripotent Stem Cells

Lineage
restricted
(committed
stem cells)

Differentiating Cells

Differentiated Cells

Equipment Utilized

Lymphoid
precursor cells

Pre-T cell

T cell

Activated T cell

Activated B cell
CFU-ML
Pre-B cell

B cell
Neutrophil

Granulocyte precursor cell
CFU-GM
Basophil
Eosinophil
Monocyte
Self-renewing
totipotent
stem cell

CFU-Meg

Myeloid
precursor cells

Macrophage

Megakaryocyte

Platelets
BFU-E
Erythroblast
RBC’s

Proerythroblast

Reticulocyte
Whole Bone Marrow
Aspirate Information
Whole Bone Marrow is drawn into a 10cc syringe containing
heparin (80U/ml of BM) from the posterior iliac crest,
25ml/site, from a maximum of four sites.
Whole Bone Marrow
• Whole Bone Marrow -> CD34+ and CD133+ isolation methods
• Small/large scale isolations
• Fluorescence staining
- Trypan blue method
- AOPI method
• Cryopreservaton Method
Post Thaw Data
Date

Tissue
Type/Unit

Lot Number

AO/PI Lot
Number

Cellometer
Count

Cellometer
Viability

Hemacytometer
Count

Trypan Blue
Viability

11-20-13 ABM-CD34+
0.5x10^6

BM4702

130904-01

0.8x10^6

97%

0.9x10^6

90%

11-20-13 ABM-CD34+
0.1x10^6

BM4699

130904-01

0.3x10^6

92%

0.3x10^6

96%
Normal Peripheral Blood
Leuko PakTM
Leuko Pak is PBMCs collected from donors by
apheresis, from Ancient Greek ἀφαίρεσις (aphairesis,
“a taking away”) is a medical technology in which the
blood of a donor or patient is passed through an
apparatus that separates out one particular
constituent and returns the remainder to the
circulation. It is thus an extracorporeal therapy. The
apparatus we us is COBE Spectra Apheresis System
manufactured by Caridian BCT. The separation of
individual blood component is done with a specialized
centrifuge. Spectra system may also be used to collect
platelets, plasma and granulocytes.
Approximately three donor blood volume is processed
and between 250-300ml of plasma and MNC is
collected/donor.
•Average cell yield: 1.3-1.5x1010
•Range: 0.9-3.5x1010
•Lead Time: 2 weeks
Normal Peripheral Blood
• NPB -> T Cells (untouched) isolation methods
• -/+ selection
• Fluorescence staining
- Trypan blue method
- AOPI method

• Cellometer method
Date

Tissue
Type/Unit

Lot Number

AO/PI Lot
Number

Cellometer
Count

Cellometer
Viability

Hemacytometer
Count

Trypan Blue
Viability

11-20-13

NPB-CD56+
5x10^6

A3860

130904-01

9.6x10^6

99%

10x10^6

96%

11-20-13

NPB-CD14+
10x10^6

A3871

130904-01

24x10^6

97%

22x10^6

91%
Custom Projects – AllCells Advantage
•
•
•
•
•
•
•
•
•

Custom tissue collection
Custom cell numbers
Custom processing/packaging
Custom aliquot and cryopreservation services
Custom cell based assays
Donor recruitment services
Multiple cell types from single donor
Donor-matched cells
Mobilized collections
Custom Assay Bioservices


Flow Cytometry Services



rare cell analysis





immunophenotyping
cell sorting

Custom Assays






Large-Scale Processing & Cell Isolation





cell cycle analysis
apoptosis, phospho protein analysis
CD34+ determination under ISHAGE guidelines

healthy or diseased human tissue
molecular products (RNA/DNA, etc.) & lysates

Drug Discovery Assay Services






Luminex® multi-analyte assays
in vitro proliferation/inhibition assays (EC50)
in vitro expansion & HSC/progenitor differentiation
toxicology studies
colony formation assays e.g. CFU-GM, CFU-Mk
Introduction to Nexcelom Bioscience, LLC
Innovation and Expertise in the Science of Cell Counting

• 10 years – design, manufacture and market cell enumeration and analysis
systems with image cytometry technologies
• Our approach: scientists help scientists to reduce error and increase assay
accuracy
• 50% employees with science degree, 8 field application scientists in US
• Work with lead labs in each research field to develop products
• Spread best practice to other labs
What is a heterogeneous cell sample?
How to select the right viability method?
Nexcelom Bioscience, LLC
Leo Chan, Ph.D. and Jean Qiu, Ph.D.
What is heterogeneous cell sample?
Heterogeneous cell sample contains target cells, other unwanted cell populations
and tissue debris.

Homogeneous
Purified CD34+ cells

Ficolled PBMCs

Whole blood
Develop better cell counting method for primary cells
• Dual fluorescence, nuclear acid binding AO/EB staining
protocol was developed at Stanford, Prof. Leonard
Herzenberg’s lab
(http://www.herzenberglab.org/Protocol-1/cell-preparation.html)

• NIH/Vaccine Research Center (VRC) used AO/EB & manual
counting
• Cellometer Vision automated AO/EB cell counting method for
NIH/VRC since 2009
• Nexcelom optimized AO/PI staining as the standard protocol
for primary cells
Dual fluorescence AO/PI viability assay
• AO is permeable to both live and dead cells
• AO binds to DNA and fluoresce bright green
• PI can only enter dead cells
- Binds to DNA of the dead cells
- Absorbs the green fluorescence of AO
- Produces bright orange / red color

• No signal is generated from non- nucleated
cells and debris
Leukapheresis samples:
over counting by bright field trypan blue method

Total cells: 119
Total FL cells: 39
Over counted cells: 80
They are not nucleated.
Over counting: 3x
The over counting ratio is sample dependent,
not a systematic trend
• Manual counting using Hemacytometer and trypan blue
• Automatic cell counting using Cellometer Vision and AO/PI
stains
• Experiment using Leukapheresis samples
Sample
Ratio
(Manual counting # to
Cellometer AO/PI #)

A

B

C

D

E

F

7X

5X

12X

3X

7X

5X
Heterogeneous cell samples mostly used
• Mouse splenocytes
• Human Peripheral Blood Mononuclear Cells
(PBMC)
• Human Mononuclear Cells (MNC)
• Human Leuko Pac
• Human Bone Marrow (BM)
• Human Cord Blood
• Mouse Tail Blood
• Bronchoalveolar lavage (BAL)
Mouse Splenocytes
BR

AOPI
Human PBMC
BR

AOPI
Human MNC
BR

AOPI
Human Leuko Pac
BR

AOPI
Human Bone Marrow
Human Cord Blood
Mouse Tail Blood
BAL
BR

AOPI
Using Trypan Blue Viability for Purified Primary Cells
AOPI stained all the CD34+ cells in BR
BR

AOPI
Which Cellometer is right for me?
• Trypan blue method is good for cell lines and purified primary
cell samples
- All Cellometer systems can perform Trypan Blue viability
measurement

• AOPI method is good for primary heterogeneous cell samples,
containing RBCs, platelets and cellular debris
- Cellometer systems with dual FL can perform AOPI viability
measurement

• Dual AOPI method was validated against total nucleated cell
counting method by lysing the RBCs
Cell Counting Requirements
•
•
•
•
•
•
•
•
•

Accuracy
Consistency
Speed
Simplicity of interface (multiple users in lab)
Minimal sample preparation
Ability to capture and store data
Create automated cell analysis reports
Avoid contact with sample (non-aerosol)
No fluidic system for potential clogging and
contamination
Benefit to Cellometer users
• Call: 978-327-5340 and you will be connected to a technical
support specialist
• Free in-lab training sessions (no limits)
• Free on-line support session via WebEx service
Nexcelom technical support specialist can connect to any Cellometer via
internet for training, trouble shooting and setting up new assays

• New in 2014: Monthly Cellometer Training Webinar
For new lab members and new applications
Q&A


Please use the prompt on the right of your screen to
send a question to the organizer



Questions will be read and answered



Any offline questions may be sent to
sales@allcells.com or sales@nexcelom.com

www.allcells.com/technical-support/
www.allcells.com/support/webinars/

www.nexcelom.com/technical-support/
www.nexcelom.com



Thank you!
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Isolating Primary Cells 101

  • 1. Isolating Primary Cells 101: Which viability method is best for your research? Ask the Experts Series #1 AllCells - Nexcelom Bioscience Exclusive Webinar Event 2/13/2014 James Lee and Leo Chan, Ph.D.
  • 2. Company Information • Provider of image cytometry technology for cell concentration and viability • Leo Chan, Ph.D. • Provider of primary cells & bioservices for pre-clinical research • James Lee
  • 3. Important Questions to Address • What is the process used for obtaining cells from bone marrow? • What is the process used for obtaining cells from peripheral blood? • What is the best and most accurate cell counting and viability method for primary cells? • How do we select the appropriate method for viability measurement?
  • 4. Key Points to Take Away • AllCells key takeaways: decrease variability, increase speed and accuracy • Nexcelom Bioscience key takeaways: the best method for concentration and viability measurement using different Cellometers, which is cell sample dependent
  • 5. Company Information ALLCELLS is a leading B2B provider of hematology-based cells & services to the world’s life science research community for the past 16 years.  Products: >800 healthy & diseased human tissue and primary cell products that enable drug discovery, stem cell research, and advancement of therapies  Bioservices: Applications of hematology / immunology expertise; cell-based assay services and customized research studies  Core Competencies: Hematology, immunology, human tissue procurement (healthy & diseased), large-scale cell isolations & characterization, stem cell expansion & differentiation  Operations: Alameda, CA (HQ), IRB approved collection programs, Tissue Bank and Biologics Licenses
  • 6. Primary Cells - Uses • • • • • Drug Development – Pre-clinical Development Predictive Toxicology Stem Cell Research/Development Regenerative Medicine Medical Device Validation
  • 7. Primary Human Cells by AllCells • Normal Peripheral Blood -> MNC -> T Cells (untouched) • Bone Marrow -> MNC -> CD34+ (organ>isolation steps-> cell types) (Cell viability, counting methods suitable for each step)
  • 8. Equipment Utilized Miltenyi’s Separation Units CliniMACS Prodigy AutoMACS Pro Separator CliniMACS Plus
  • 9. Pluripotent Stem Cells Lineage restricted (committed stem cells) Differentiating Cells Differentiated Cells Equipment Utilized Lymphoid precursor cells Pre-T cell T cell Activated T cell Activated B cell CFU-ML Pre-B cell B cell Neutrophil Granulocyte precursor cell CFU-GM Basophil Eosinophil Monocyte Self-renewing totipotent stem cell CFU-Meg Myeloid precursor cells Macrophage Megakaryocyte Platelets BFU-E Erythroblast RBC’s Proerythroblast Reticulocyte
  • 10. Whole Bone Marrow Aspirate Information Whole Bone Marrow is drawn into a 10cc syringe containing heparin (80U/ml of BM) from the posterior iliac crest, 25ml/site, from a maximum of four sites.
  • 11. Whole Bone Marrow • Whole Bone Marrow -> CD34+ and CD133+ isolation methods • Small/large scale isolations • Fluorescence staining - Trypan blue method - AOPI method • Cryopreservaton Method Post Thaw Data Date Tissue Type/Unit Lot Number AO/PI Lot Number Cellometer Count Cellometer Viability Hemacytometer Count Trypan Blue Viability 11-20-13 ABM-CD34+ 0.5x10^6 BM4702 130904-01 0.8x10^6 97% 0.9x10^6 90% 11-20-13 ABM-CD34+ 0.1x10^6 BM4699 130904-01 0.3x10^6 92% 0.3x10^6 96%
  • 12. Normal Peripheral Blood Leuko PakTM Leuko Pak is PBMCs collected from donors by apheresis, from Ancient Greek ἀφαίρεσις (aphairesis, “a taking away”) is a medical technology in which the blood of a donor or patient is passed through an apparatus that separates out one particular constituent and returns the remainder to the circulation. It is thus an extracorporeal therapy. The apparatus we us is COBE Spectra Apheresis System manufactured by Caridian BCT. The separation of individual blood component is done with a specialized centrifuge. Spectra system may also be used to collect platelets, plasma and granulocytes. Approximately three donor blood volume is processed and between 250-300ml of plasma and MNC is collected/donor. •Average cell yield: 1.3-1.5x1010 •Range: 0.9-3.5x1010 •Lead Time: 2 weeks
  • 13. Normal Peripheral Blood • NPB -> T Cells (untouched) isolation methods • -/+ selection • Fluorescence staining - Trypan blue method - AOPI method • Cellometer method Date Tissue Type/Unit Lot Number AO/PI Lot Number Cellometer Count Cellometer Viability Hemacytometer Count Trypan Blue Viability 11-20-13 NPB-CD56+ 5x10^6 A3860 130904-01 9.6x10^6 99% 10x10^6 96% 11-20-13 NPB-CD14+ 10x10^6 A3871 130904-01 24x10^6 97% 22x10^6 91%
  • 14. Custom Projects – AllCells Advantage • • • • • • • • • Custom tissue collection Custom cell numbers Custom processing/packaging Custom aliquot and cryopreservation services Custom cell based assays Donor recruitment services Multiple cell types from single donor Donor-matched cells Mobilized collections
  • 15. Custom Assay Bioservices  Flow Cytometry Services   rare cell analysis   immunophenotyping cell sorting Custom Assays     Large-Scale Processing & Cell Isolation    cell cycle analysis apoptosis, phospho protein analysis CD34+ determination under ISHAGE guidelines healthy or diseased human tissue molecular products (RNA/DNA, etc.) & lysates Drug Discovery Assay Services      Luminex® multi-analyte assays in vitro proliferation/inhibition assays (EC50) in vitro expansion & HSC/progenitor differentiation toxicology studies colony formation assays e.g. CFU-GM, CFU-Mk
  • 16. Introduction to Nexcelom Bioscience, LLC Innovation and Expertise in the Science of Cell Counting • 10 years – design, manufacture and market cell enumeration and analysis systems with image cytometry technologies • Our approach: scientists help scientists to reduce error and increase assay accuracy • 50% employees with science degree, 8 field application scientists in US • Work with lead labs in each research field to develop products • Spread best practice to other labs
  • 17. What is a heterogeneous cell sample? How to select the right viability method? Nexcelom Bioscience, LLC Leo Chan, Ph.D. and Jean Qiu, Ph.D.
  • 18. What is heterogeneous cell sample? Heterogeneous cell sample contains target cells, other unwanted cell populations and tissue debris. Homogeneous Purified CD34+ cells Ficolled PBMCs Whole blood
  • 19. Develop better cell counting method for primary cells • Dual fluorescence, nuclear acid binding AO/EB staining protocol was developed at Stanford, Prof. Leonard Herzenberg’s lab (http://www.herzenberglab.org/Protocol-1/cell-preparation.html) • NIH/Vaccine Research Center (VRC) used AO/EB & manual counting • Cellometer Vision automated AO/EB cell counting method for NIH/VRC since 2009 • Nexcelom optimized AO/PI staining as the standard protocol for primary cells
  • 20. Dual fluorescence AO/PI viability assay • AO is permeable to both live and dead cells • AO binds to DNA and fluoresce bright green • PI can only enter dead cells - Binds to DNA of the dead cells - Absorbs the green fluorescence of AO - Produces bright orange / red color • No signal is generated from non- nucleated cells and debris
  • 21. Leukapheresis samples: over counting by bright field trypan blue method Total cells: 119 Total FL cells: 39 Over counted cells: 80 They are not nucleated. Over counting: 3x
  • 22. The over counting ratio is sample dependent, not a systematic trend • Manual counting using Hemacytometer and trypan blue • Automatic cell counting using Cellometer Vision and AO/PI stains • Experiment using Leukapheresis samples Sample Ratio (Manual counting # to Cellometer AO/PI #) A B C D E F 7X 5X 12X 3X 7X 5X
  • 23. Heterogeneous cell samples mostly used • Mouse splenocytes • Human Peripheral Blood Mononuclear Cells (PBMC) • Human Mononuclear Cells (MNC) • Human Leuko Pac • Human Bone Marrow (BM) • Human Cord Blood • Mouse Tail Blood • Bronchoalveolar lavage (BAL)
  • 32. Using Trypan Blue Viability for Purified Primary Cells AOPI stained all the CD34+ cells in BR BR AOPI
  • 33. Which Cellometer is right for me? • Trypan blue method is good for cell lines and purified primary cell samples - All Cellometer systems can perform Trypan Blue viability measurement • AOPI method is good for primary heterogeneous cell samples, containing RBCs, platelets and cellular debris - Cellometer systems with dual FL can perform AOPI viability measurement • Dual AOPI method was validated against total nucleated cell counting method by lysing the RBCs
  • 34. Cell Counting Requirements • • • • • • • • • Accuracy Consistency Speed Simplicity of interface (multiple users in lab) Minimal sample preparation Ability to capture and store data Create automated cell analysis reports Avoid contact with sample (non-aerosol) No fluidic system for potential clogging and contamination
  • 35. Benefit to Cellometer users • Call: 978-327-5340 and you will be connected to a technical support specialist • Free in-lab training sessions (no limits) • Free on-line support session via WebEx service Nexcelom technical support specialist can connect to any Cellometer via internet for training, trouble shooting and setting up new assays • New in 2014: Monthly Cellometer Training Webinar For new lab members and new applications
  • 36. Q&A  Please use the prompt on the right of your screen to send a question to the organizer  Questions will be read and answered  Any offline questions may be sent to sales@allcells.com or sales@nexcelom.com www.allcells.com/technical-support/ www.allcells.com/support/webinars/ www.nexcelom.com/technical-support/ www.nexcelom.com  Thank you!