Regulatory Science Considerations 

for Cell Counting

Steven R. Bauer, Ph.D.

US Food and Drug Administration

Center for Biologics Evaluation and Research

Office of Tissues and Advanced Therapies

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Cell Counting is Crucial for Products

in Multiple FDA Centers

• CBER: many cell-based products, cell-based assays

• CDER: stem-cell based screening assays for drug 

development, cell-based production of biologics 

• CDRH: devices for counting cells, diagnostics

• CVM: many cell-based products

• CFSAN: presence of cells in certain products

• My examples: CBER- regulated cell-based products

• Generally applicable to other FDA-regulated products

2
Regulatory Considerations for All 

Biologics

• Safety, efficacy, purity, potency, identity, 

quality

• Oversight of both product and process

• Quality control of source materials, 

intermediates, and product

• Reproducibility of lots

• Comparability after manufacturing change

www.fda.gov

Reliable cell count is crucial to all of these!

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Clinical

Response

control

What do you 

measure to identify 

the effective 

products?

Nothing

active

Marginal 

effect

Total cure

Slide courtesy of Malcolm Moos 4

Heterogeneity

of cells

Diversity of 

patients
Can we develop ways to identify 

Quality Attributes that

predict safety and effectiveness?

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Developmental Stages

Environmental Influences

Selfrenewal Commitment DifferentiationTerminal

Differentiation

How Can We Help Fulfill the 

Tremendous Promise?

(Purity, Identity, Potency)
Counting is Crucial During All Stages 

of Cell-based Product Development

www.fda.gov

IND BLA

Development Preclinical Phase I Phase II Phase III Marketing

Safety

Quality

Pre-IND

IND Submitted BLA Submitted

Evaluation of safety & efficacy

Critical Path

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Analytical Method Development

Pre-Clinical

• Selection

•Development

•Optimization

Phase I

•Methods 

qualified

• Set tentative 

release/stability 

criteria

Phase II

•Assay 

optimization

• Consider 

validation 

acceptance 

criteria

•Delineate/initiate 

assay validation 

parameters

Phase III and 

BLA

• Full assay 

validation by 

licensure 

(recommended 

by Phase III)

Assay Qualification: Determining if an assay is suitable for its 

intended purpose

Assay Validation: Assuring that the assay is suitable to its intended 

purpose on a routine basis 5
Cell Product Characterization

Cell count/viability

Purity 

 Free of extraneous 

materials 

 Identity 

 Specific test to 

distinguish it from others 

Potency 

 Assay for biological 

function

• Sponsor determined methods

• Cell count crucial for many of 

these parameters

• Preclinical safety studies and 

clinical dose based on these 

tests

• Viability recommendation: 

70% 

– When are you counting?

– Thawed or “recovered”?

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Additional Considerations for 

Cell-based Products 

• Counting often established for one product by one 

manufacturer in one site

• Other considerations

– Multiple clinical sites

– Multiple manufacturing sites

– Technology Transfer (new site, scale out, etc.)

– Influence of cell delivery method

– Automated QC methods

• Standards (written, physical) would be beneficial

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Validation Considerations

• Useful references

– Analytical Procedures and Methods Validation for Drugs and Biologics. Guidance for Industry 2015.

• For INDs, sufficient information is required at each phase of an investigation to ensure proper identity, quality, 

purity, strength, and/or potency. The amount of information on analytical procedures and methods suitability 

will vary with the phase of the investigation.

– ICH VALIDATION OF ANALYTICAL PROCEDURES: TEXT AND METHODOLOGY Q2(R1) 

• Examples for cell counting

– Robustness/ruggedness

• Capacity to remain unaffected by small, deliberate variations in method to indicate reliability during normal use

– Linearity

• Dilution series and curve fitting (R2) over range tested

– Accuracy

• Variability (%CV) across replicate samples, multiple dilutions, (different methods)

– Range

• Cell dilutions that are accurate and precise

– Intermediate precision

• Different lots, different operators, different days

– Precision: repeatability

• Identical cell lots in same laboratories, same operators, same apparatus, short time frame

– LOD

• Lowest cell density detected in serial dilutions

– LOQ

• Lowest cell density that is accurate and precise

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MSCs and Product Characterization

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Adherent in culture

CD73+, 90+, 105+

Lymphocyte marker negative

Manufactured

MSC

Osteoblasts

Adipocytes

Chondrocytes

Immunomodulatory/

Anti-Inflammatory

Mesenchymal 

Stem Cell ?
Heterogeneity of Cells

– Total count of cells may not = total biological 

activity

• Not all CD34+ cells are long-term HSCs!

• Not all MSCs have the same biological activities

PCBM1632

110877167696

PCBM1641PCBM1662

8F3560

127756

PCBM1655

0

2

4

6

8

10

12

14

16

Passage 3

Passage 5

Passage 7

% Nile Red Positive

MSC Adipogenic Activity 

Decreases with Tissue 

Culture Passage: Donor 

Differences

Lo Surdo JL et al., Cytotherapy, 2013.

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Benefits of Standards

(Written, Physical)

• Confidence/comparison

• Basic and Translational Research 

– Academic labs

– R&D labs

– Within context of one sponsor

– Within community of developers

• Facilitate development of safe and effective cellbased products

– Precise and Accurate Cell Counts

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OTAT Contact Information

Steven R. Bauer, Ph.D. 

steven.bauer@fda.hhs.gov

240-402-9385

Regulatory Questions: 

Contact the Regulatory Management Staff in OTAT at 

OTATRPMS@fda.hhs.gov

or Lori.Tull@fda.hhs.gov 

or by calling (240) 402-8361

OCTGT Learn Webinar Series:

http://www.fda.gov/BiologicsBloodVaccines/NewsEvents/ucm

232821.htm 

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Public Access to CBER

CBER website:

http://www.fda.gov/BiologicsBloodVaccines/default.htm

Phone: 1-800-835-4709 or 240-402-8010

Consumer Affairs Branch (CAB) 

Email: ocod@fda.hhs.gov

Phone: 240-402-8010

Manufacturers Assistance and Technical Training Branch (MATTB)

Email: industry.biologics@fda.gov

Phone: 240-402-8010

Follow us on Twitter 

https://www.twitter.com/fdacber

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