CELL COUNTING WORKSHOP NIST

CELLCOUNTINGWORKSHOPNIST

PhilipMathuisApril10,2017

FROMIMAGETOACTION

2STRICTLYCONFIDENTIAL

ANALYZEIDENTIFYCOUNTVIABILITY

PATENTS

DIAGNOSTICSUPSIDE

PEOPLEANDECOSYSTEM

FUNDING

BEACHHEADMARKETBIOPROCESSING

BACKGROUND

16OVIZIO RESEARCHECOSYSTEM

GRANTSEQUITY

4.700K€15.211K€

SUSPENSIONUNITSADHERENTUNITS

6

OTHERPARTNERSAPPLIKONPALLLIFESCIENCES

FOUNDEDINDEC2009SPIN-OFFULBISO13485INPROGRESS

TOTALMARKET:3,46B$TOTAL#OFTESTS:200MCOSTPERSMEAR:18– 30$GROWTHRATE:4%

TOTALMARKET:1,1B$GROWTHRATE:6%

PAPSMEARSINUNITS

5.801

28.520

54

65

63

18 EUROPE

USA

ASIA

ROW

3

STRICTLYCONFIDENTIAL

7

1

ULBGRANTEDOVIZIOFILEDOVIZIOGRANTED

DDHM technology


PatentedviabilitymethodInventedbyprof.FrankDuboisULB(patentUS7,362,449&ep1631788)

INTENSITYIMAGE PHASEIMAGE

3DTOPVIEWIMAGE 3DIMAGE

REFOCUSSING

290CELLS– 95%CONF.

5

HOWDOWEANALYZEIMAGES


ACQUIREHOLOGRAM

COMPUTEPHASE

DETECTLIGHTCONES

REFOCUSALLOBJECTS

EXTRACTFINGERPRINT

HOLOGRAPHICFINGERPRINTFORCELLS


74PARAMETERSARERECORDEDPERCELL– MACHINELEARNING

VOLUME

DIAMETER

SIZE

CIRCULARITY

LENSBEHAVIOR

HEIGHTVARIANCE

MEMBRANEINTEGRITY

NUCLEUSTOCYTOPLASMRATIO

CELLPERIMETER

• IDENTIFY• DIFFERENTIATE• COUNT• COMPARE• ANALYZE• ASSESSVIABILITY

PARAMETERSSYSTEMMORPHOLOGYOPTICALPHASETEXTUREINTENSITYTEXTURE

MACHINELEARNINGMETHODOLOGY


STEP1LEARNINGSETCREATION

STEP2SYSTEMTRAINING

STEP3PREDICTION

100%LIVINGCELL

100%DEADCELLS

FEEDSYSTEMWITHKNOWNMIXTURES

FROM100%TO0%INSTEPSOF10



Proprietary&Confidential©2016Ovizio,AllRightsReserved 9

BENEFITS OF AUTOMATION

• INCREASEDCONTROL

• TIMEGAINANDTRACEABILITYOFRESULTS

• INCREASEDREPRODUCIBILITY

• DRASTICREDUCTIONOFMANUALOPERATIONS

• REDUCEDINVESTMENTANDFTECOST– FASTROI

OSONE:CELLVIABILITYINDICATORS


OSONE:SINGLECELLANALYSES


OSONE:WEBINTERFACE


CONTINUOUSMONITORING


STATISTICALRELEVANCEILINEF:6000IMAGESPERDAYCELLCOUNTER:50-100IMAGESPERSAMPLE

NODILLUTIONREQUIRED


OPC Integration

TECHNOLOGYCOMPARED


R=0,968

VARIABILITY-TCD


y=0.9771xR²=0.99007

02468

10121416

0 2 4 6 8 10 12 14 16

TCD-B

io-Con

nect2(1

0E+6cells/m

l)

TCD- BioConnect1(10E+6cells/ml)

TCD- COMPARISONy=1.0147xR²=0.98705

0

20

40

60

80

100

120

0 20 40 60 80 100 120Viab

ility-Bio-Co

nnect2

(%)

Viability- BioConnect1(%)

VIABILITYCOMPARISON

y=0.9933xR²=0.99651

0

2

4

6

8

10

12

14

0 2 4 6 8 10 12 14

VCD-B

io-Con

nect(1

0E+6cells/m

l)

VCD- BioConnect1(10E+6cells/ml)

VCDCOMPARISON INSAMPLE

CASESTUDY:VIRALLOADMEASUREMENT


PROTEINEXPRESSION

INFECTEDCELL

NON-INFECTEDCELL


CASESTUDY – DISCRIMINATECELLTYPES

• USCUSTOMER• IMMUNOTHERAPY• ENHANCED

DENDRITICCELLS• 6– DAYPROCESS

BACKGROUND

• IN-PROCESSCOUNTINGOFVIABLECELLDENSITYOF:• REDBLOODCELLS• GRANULOCYTES• LYMPHOCYTES• DENDRITICCELLS

• NOLOSSOFSAMPLEVOLUME

CHALLENGES

CRITICALPARAMETERSHEREARETOMONITORCELLCHARACTERISTICS


CASESTUDY– RESULTSCOUNT

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

OVIZIO FCM

6,5MCells/ml

Monocytes Lymphocytes Granulocytes

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

OVIZIO FCM

0,79MCells/ml

Monocytes Lymphocytes Granulocytes

MULTILAYERRECIPIENTS


MSCCELLS– XP10

MULTIPLATEOBSERVATION

XP10_Day5_plate1 XP10_Day5_plate3 XP10_Day5_plate6 XP10_Day5_plate8

Plate1 Plate3 Plate6 Plate8


CLIENTTEST:AUTOMATEDCONFLUENCY

23

20130205ClientMSC StemCells –70%


CLIENTTEST:AUTOMATEDCONFLUENCY

24

20130205ClientMSC StemCells –90%


OVIZIOVS MANUALMEASUREMENT

25

CONFLUENCEMEASUREMENTSMATCHTHEESTIMATIONSOFAHUMANOPERATORCLOSELY.CONFLUENCEISTHEPARAMETEROFMAINIMPORTANCEATTHISSTAGE.


y=0.8417x+9.9266R²=0.89186

50

55

60

65

70

75

80

85

90

95

100

50 60 70 80 90 100

othercaptures

E116-MADCS2-1-J4

manualmeasurementoftheconfluence (%)

confluence

inOsO

ne(%)


THANKYOUFORYOURATTENTION!Wewouldlovetocontinuethediscussionwithyou…

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CELL COUNTING WORKSHOP NIST

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