CELL	COUNTING	WORKSHOP

NIST

Philip	Mathuis	April	10,	2017
FROM	IMAGE	TO	ACTION

2

STRICTLY	CONFIDENTIAL

ANALYZE

IDENTIFY

COUNT

VIABILITY
PATENTS

DIAGNOSTICS	UPSIDE

PEOPLE	AND	ECOSYSTEM

FUNDING

BEACHHEAD	MARKET

BIOPROCESSING

BACKGROUND

16	OVIZIO RESEARCH	ECOSYSTEM

GRANTS

EQUITY

15.211	K€ 4.700	K€

SUSPENSION	UNITS

ADHERENT	UNITS

6

PARTNERS OTHER

APPLIKON

PALL	LIFE	SCIENCES

FOUNDED	IN	DEC	2009

SPIN-OFF	ULB

ISO	13485	IN	PROGRESS

TOTAL	MARKET:	3,46B$

TOTAL	#	OF	TESTS:	200	M

COST	PER	SMEAR:	18	– 30$

GROWTH	RATE:	4%

TOTAL	MARKET:	1,1	B$

GROWTH	RATE:	6%

PAP	SMEARS	IN	UNITS

5.801

28.520

54

65

63

18 EUROPE

USA

ASIA

ROW

3

STRICTLY	CONFIDENTIAL

7

1

ULB	GRANTED

OVIZIO	FILED

OVIZIO	GRANTED
DDHM technology

4

STRICTLY	CONFIDENTIAL

Patented	viability	method

Invented	by	prof.	Frank	Dubois	ULB	(patent	US		7,362,449	&	ep1631788)	
INTENSITY	IMAGE PHASE	IMAGE

3D	TOP	VIEW	IMAGE 3D	IMAGE

REFOCUSSING

290	CELLS	– 95%	CONF.

5
HOW	DO	WE	ANALYZE	IMAGES

6

STRICTLY	CONFIDENTIAL

ACQUIRE	

HOLOGRAM

COMPUTE	

PHASE

DETECT	

LIGHT	CONES

REFOCUS	ALL	

OBJECTS

EXTRACT	

FINGERPRINT
HOLOGRAPHIC	FINGERPRINT	FOR	CELLS

7

STRICTLY	CONFIDENTIAL

74	PARAMETERS	ARE	RECORDED	PER	CELL	– MACHINE	LEARNING

VOLUME

DIAMETER

SIZE

CIRCULARITY

LENS	

BEHAVIOR

HEIGHT	

VARIANCE

MEMBRANE	

INTEGRITY

NUCLEUS	TO	

CYTOPLASM	

RATIO

CELL	

PERIMETER

• IDENTIFY

• DIFFERENTIATE

• COUNT

• COMPARE

• ANALYZE

• ASSESS	VIABILITY

PARAMETERS

SYSTEM

MORPHOLOGY

OPTICAL

PHASE	TEXTURE

INTENSITY	TEXTURE
MACHINE	LEARNING	METHODOLOGY

8

STRICTLY	CONFIDENTIAL

STEP	1

LEARNING	SET	CREATION

STEP	2

SYSTEM	TRAINING

STEP	3

PREDICTION

100	%	LIVING	CELL

100%	DEAD	CELLS

FEED	SYSTEM	WITH	

KNOWN	MIXTURES

FROM	100%	TO	0%		

IN	STEPS	OF	10

STEP	1

LEARNING	SET	CREATION

STEP	1

LEARNING	SET	CREATION
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BENEFITS OF AUTOMATION

• INCREASED	CONTROL	

• TIME	GAIN	AND	TRACEABILITY	OF	

RESULTS

• INCREASED	REPRODUCIBILITY	

• DRASTIC	REDUCTION	OF	MANUAL	

OPERATIONS	

• REDUCED	INVESTMENT	AND	FTE	COST	

– FAST	ROI
OSONE:	CELL	VIABILITY	INDICATORS

10

STRICTLY	CONFIDENTIAL
OSONE:	SINGLE	CELL	ANALYSES

11

STRICTLY	CONFIDENTIAL
OSONE:	WEB	INTERFACE

12

STRICTLY	CONFIDENTIAL
CONTINUOUS	MONITORING

13

STRICTLY	CONFIDENTIAL

STATISTICAL	RELEVANCE

ILINE	F:	6000	IMAGES	PER	

DAY

CELL	COUNTER:	50-100	

IMAGES	PER	SAMPLE

NO	DILLUTION	REQUIRED
Proprietary	&	Confidential	©	2016	Ovizio,	All	Rights	Reserved 14

OPC Integration
TECHNOLOGY	COMPARED

15

STRICTLY	CONFIDENTIAL

R=	0,968
VARIABILITY-TCD

16

STRICTLY	CONFIDENTIAL

y	=	0.9771x

R2	=	0.99007

0

2

4

6

8

10

12

14

16

0 2 4 6 8 10 12 14 16

TCD	- Bio-Connect	2	(10E+6	cells/ml)

TCD	- BioConnect	1	(10E+6	cells/ml)

TCD	- COMPARISON

y	=	1.0147x

R2	=	0.98705

0

20

40

60

80

100

120

Viability	

0 20 40 60 80 100 120

- Bio-Connect	2	(%)

Viability- BioConnect	1	(%)

VIABILITY	COMPARISON

y	=	0.9933x

R2	=	0.99651

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

VCD	- Bio-Connect	(10E+6	cells/ml)

VCD	- BioConnect	1	(10E+6	cells/ml)

VCD	COMPARISON IN	SAMPLE
CASE	STUDY:	VIRAL	LOAD	MEASUREMENT

17

STRICTLY	CONFIDENTIAL

PROTEIN	

EXPRESSION

INFECTED	

CELL

NONINFECTED	

CELL
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CASE	STUDY – DISCRIMINATE	CELL	TYPES

• US	CUSTOMER

• IMMUNOTHERAPY

• ENHANCED	

DENDRITIC	CELLS

• 6	– DAY	PROCESS

BACKGROUND

• IN-PROCESS	COUNTING	OF	VIABLE	

CELL	DENSITY	OF:

• RED	BLOOD	CELLS

• GRANULOCYTES

• LYMPHOCYTES

• DENDRITIC	CELLS

• NO	LOSS	OF	SAMPLE	VOLUME

CHALLENGES

CRITICAL	PARAMETERS	HERE	ARE	TO	MONITOR	CELL	CHARACTERISTICS	
CELL	THERAPY	CLOSED	MANUFACTURING	SYSTEM	DENDRIDIC	CELLS

Proprietary	&	Confidential	©	2016	Ovizio,	All	Rights	 19

Reserved

MONOCYTES MDC’s LYMPHOCYTES GRANULOCYTES RBC’S
Proprietary	&	Confidential	©	2016	Ovizio,	All	Rights	Reserved 20

CASE	STUDY	– RESULTS	COUNT

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

OVIZIO FCM

6,5	M	Cells/ml

Monocytes Lymphocytes Granulocytes

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

OVIZIO FCM

0,79	M	Cells/ml

Monocytes Lymphocytes Granulocytes
MULTI	LAYER	RECIPIENTS

21

STRICTLY	CONFIDENTIAL
MSC	CELLS	– XP10

MULTIPLATE	OBSERVATION

XP10_Day5_plate	1 XP10_Day5_plate	3 XP10_Day5_plate	6 XP10_Day5_plate	8

Plate	1 Plate	3 Plate	6 Plate	8

22

STRICTLY	CONFIDENTIAL
CLIENT	TEST:	AUTOMATED	CONFLUENCY

23

20130205	Client	MSC Stem	Cells –70%

STRICTLY	CONFIDENTIAL
CLIENT	TEST:	AUTOMATED	CONFLUENCY

24

20130205	Client	MSC Stem	Cells –90%

STRICTLY	CONFIDENTIAL
OVIZIO	VS MANUAL	MEASUREMENT

25

CONFLUENCE	MEASUREMENTS	MATCH	THE	ESTIMATIONS	OF	A	HUMAN	OPERATOR	

CLOSELY.	CONFLUENCE	IS	THE	PARAMETER	OF	MAIN	IMPORTANCE	AT	THIS	STAGE.

STRICTLY	CONFIDENTIAL

y	=	0.8417x	+	9.9266

R2	=	0.89186

50

55

60

65

70

75

80

85

90

95

100

50 60 70 80 90 100

other	captures

E116-MAD	CS2-1-J4	

manual	measurement	of	the	confluence (%)

confluence in	OsOne (%)
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