European Bank for induced pluripotent Stem Cells04 Poster_V05.indd 1 09.06.16 00:21 EBiSC: The Banking Process The EBiSC - European Bank for induced pluripotent Stem Cells project

European Bank for induced pluripotent Stem Cells

About EBiSCThe EBiSC Poster Book

Information on EBiSCwww.ebisc.eu Access to the EBiSC iPSC Catalogue https://cells.ebisc.org

Contact [email protected]

Follow us on Twitter @EBiSC_cells

EBiSC: Who We Are

The EBiSC - European Bank for induced pluripotent Stem Cells project has received support from the Innovati ve Medicines Initi ati ve Joint Undertaking under grant agreement n° 115582, resources of which are composed of fi nancial contributi on from the European Union‘s Seventh Framework Programme (FP7/2007-2013) and EFPIA companies’ in kind contributi on. www.imi.europa.eu

htt ps://cells.ebisc.orgwww.ebisc.eu

Project Coordinator Tim Allsopp (Pfi zer-Neusenti s, UK)

Manager of the Public PartnersAidan Courtney, (Roslin Cell Sciences, UK)

WP4.2 Training: Glyn Stacey (Nati onal Insti tute for Biological Standards and Controls-NIBSC, UK)

Work Package 5Quality Control and Characterisati on: Glyn Stacey (NIBSC, UK)

Work Package 7 WP7.1/WP7.2 Informati on Management Systems Develop-ment and Operati on Helen Parkinson (EMBL-European Bioinformati cs Insti tute, UK)

Work Package 6WP6.1 Storage and Distributi on: Bryan Bolton (European Collecti on of Cell Cultures-ECACC, UK)

WP6.2 Validati ng the End Product: Christi an Clausen (Bioneer, Denmark)

Work Package 1WP1.1 Governance and Policy WP1.3 Communicati on and Disseminati on: Aidan Courtney (Roslin Cell Sciences, UK) and Beate Kreisel (ARTTIC, France)WP1.2 Assessing the Market as a whole

Work Package 2WP2.1 Procurement and Sustainability of iPSC line producti on: Paul de Sousa (Roslin Cell Sciences, UK)

WP2.2 Central & Secondary iPSC Faciliti es: Julie Holder (Roslin Cell Sciences, UK)

Work Package 3Bio-engineering and Automati on: Heiko Zimmermann, (Fraunhofer Insti tute for Biomedical Engineering, Germany)

Work Package 4WP4.1 Ethics & Engagement: Responsible Research & Innovati on: Carol George and Shawn Harmon (University of Edinburgh, UK)

EFPIA companies SME‘s Universiti es, research organisati ons, public bodies, non-profi t groups

01 Poster_V05.indd 1 10.06.16 16:58

EBiSC: Project Timeline


Jan 2014Project kick-off

March 2014First ‘benchmark’ protocols for cell processing distributed to iPSC depositor centres

May 2014First EBiSC Cell Processing and Quality Control training course and workshop delivered

June 2014• First Hot Start lines from Roslin Cell

Sciences begin expansion in Edinburgh

• Standardised format for cell line nomenclature, tracking and labelling agreed with all parti es in Edinburgh

End 2019Self-sustaining organisati on realised


Mar 2015Cell processing and QC initi ated at Babraham Central Facility

July 2015Performance Qualifi cati on of Babraham Central Facility complete. Ready for EBiSC producti on.

Dec 2016Integrati on of develop-ment acti viti es for pro-cess opti misati on

July 2014• Standard Operati ng Procedures

and Forms for iPSC line producti on approved

• First commissioning of new iPSC lines for the Foundati onal Collecti on is approved

Aug 2014• University of Newcastle

become the fi rst EBiSC depositor to create and ship a cell line to Edinburgh facility

• EBiSC demonstrates ability to re-consent donors of existi ng iPSC lines to enable their depositi on in the bank

Oct 2014First iPSC lines deposited from non-consorti um partners

November 2016EBiSC training course on reprogramming

2014

Nov 2014The lease for the Bab-raham Central Facility is agreed and the fi rst employee is recruited

Dec 2014• Gap analysis completed

to identi fy target diseases for development of EBiSC catalogue beyond the Foundati onal Collecti on

• Consorti um Material Deposit Agreement eff ecti ve

• EBiSC distributes its fi rst cell lines to Academic and EFPIA industry partners thus demonstrati ng end-to-end capacity of EBiSC operati on

• EBiSC practi cal training course on hiPSC culturing

Jan 2015Fraunhofer-IBMT proving lab established at Babraham

June 2016ISSCR 2016 Annual Meeti ng in San Francisco: EBiSC Focus Session and Meet-up Hub

2015

October 2016 EBiSC booth at the ESGCT/ISSCR Inter-nati onal Symposium Florence

March 2016 EBiSC Catalogue Launch,htt ps://cells.ebisc.org

June 2015ISSCR 2015 Annual Meeti ng in Stockholm: EBiSC Meet-up Hub

2016

2019

May 2016EBiSC practi cal training course in iPSC culture, preservati on, QC and diff erenti ati on

02 Poster_V04.indd 1 09.06.16 20:42

EBiSC: Automati on Strategies for the Expansion and Cryostorage of iPS cells in EBiSC


Establishment of EBiSC MirrorStorage Facility (Sulzbach, Germany)

Establishment „Automati on Enabling Lab“ (Babraham, UK)Automated Technologies For Large-Scale Expansion

Establishment of Effi cient Cryopreservati on ProceduresSurface-based vitrifi cati on of hPSCs:

• High viability and recovery• Maintained pluripotency• Ready-to-use• No ti me delay aft er thawing

Automated pipetti ng platf orm Freedom EVO200TM from TECAN adapted to hiPSC-expansion:• Highly fl exible and complex system• High-throughput capacity with up to 12x Robofl ask (93 cm2)

passaging per 8h• Complicated and ti me consuming handling • Not recommended for complete expansion workfl ows

BiolevitatorTM and LevitubesTM

from Hamilton:• Robust and reproducible• Volume of 4x50ml• Low shear forces• No upscaling possible• Further evaluati on necessary

The HexaBatchTM system from HexaScreen Culture Technologies:• Robust and reproducible• Volume of 6x15ml• Medium shear forces• No upscaling possible• Not recommended for EBiSC

Addressing the aims of EBiSC:• Adaptati on of current protocols for scalable

iPSC expansion• Identi fi cati on and installati on of automati on

for scalable expansion of iPSC

• Establishment of innovati ve automated technology as e.g. closed cold chain and reliable sample identi fi cati on

• Establishment of reliable biobanking infrastructure

• Establishment of effi cient, standardised and reproducible protocols of high quality cryopreservati on of iPSC

The automated cell culture system CompacT SelecTTM from TAP Biosystems:• Highly reproducible workfl ow• Hosti ng of up to 90 T175 fl asks, automated

passaging of cells 48 fl asks/24h• Easy and fast handling• Recommended for EBiSC

Automated cell culture platf orms

Bioreactors

MicroLAB StarTM from Hamilton (@UK Bonn):• Reliable processing with high reproducibility for walkaway

lab automati on• Media changes, cell seeding and coati ng in MTP (limited to MTP)• Comprehensive handling due to high system variability• Recommended for EBiSC (for small/medium scale cell producti on)

www.hexascreen.com

www.biolevitator.com

www.hamiltoncompany.com

www.askion.com

Magneti c sti rrer and spinner fl asks:• Robust and reproducible• Volume up to several litres• High shear forces• No sensors, not automated• Not recommended for EBiSC

www.2mag.de

C-lineTM system from Askion:

• Fully automated sample storage for closed cold chain • Storage below -150°C• Working temperature -80 to -110°C


03 Poster_V04.indd 1 09.06.16 21:13

EBiSC: Data Management Overview


The EBiSC Information Management System (IMS) seamlessly connects the flow of data across the entire supply chain from cell line deposition through to customer order and supply. It provides a wealth of descriptive information on the cell lines and will also capture user generated data to enrich and add value to a constantly expanding knowledge base for every cell line in the bank.

The IMS provides a web-based platf orm for ease of access that maximises the uti lity of the EBiSC resource and provides an effi cient facility for purchasing cell lines quickly and at reasonable cost.

The IMS will also support the integrati on of usergenerated content on cell lines, accruing substanti alinformati on over ti me and enabling subsequent users access to cell lines of intrinsically greater value.

EBiSC data management elements

hPSCreg

EBiSC Informati on Management System (IMS)

• Online registry of human pluripotent cell lines

• EBiSC cell line data

• BioSamples database• Unique data /

sample descripti ons• Linkage to data sets• Data management

• LabWare LIMS• Cell line processing• Cell line QC• Producti on intelligence

• Cell line distributi on• E-commerce

EBI Central Facility ECACC

Depositor Management

E-commerce

Customer Web-portal

Order management

Cell line batch informati on

Clinical data

Inventory

Ethical provenance

Publicati on tracking

Knowledge base

management

Diverse range of external databases housing cell line

characterisati on and omics dataPublicati ons

User generated

data

Cells to users with an enhanced data package


Initi al data package from

depositor

04 Poster_V05.indd 1 09.06.16 00:21

EBiSC: The Banking Process


Cell line receipt and registrati on

Thawing of cell line

1 day

Culture in quaranti ne

1 weekExpansion phase

2 weeks

Harvest and Banking

1 day

Post Bank expansion checks

2 weeks

Bank storage ready

for distributi on

Process steps

Cell Line Received

Cell LineRegistered

into cryostorage

Thaw into 2 wells

(80:20% split) in 6 well plate and incubate

Passage x3 on Vitronecti n or Geltrex in

anti bioti c freemedia

Harvest 60 wells or 4 x T150

Thaw 1 vial into 2 wells

Hold in central facility

cryo store

Expand to 4 wells

Prepare sam-ples for fi nal QC

Checks

Prepare certi fi cate of

analysis

Prepare samples for fi nal

QC checks

Expand into

4 wells

Expand into

16 wells

Expand into T75

fl ask

Expand into

60 wells

Expand into 4x

T150 fl ask

EBiSC/SOP/1 EBiSC/SOP/2SOP/EQP/75

EBiSC/FRM/1EBiSC/SOP/10EBiSC/FRM/3

EBiSC/SOP/9EBiSC/FRM/4

EBiSC/SOP/8



Mycoplasma Free

All QC Checks complete

Final stage

distribu-ti on atECACC

Mirror bank at Fraun-hofer

(IMBT)

In process control checks

ViabilityGeneti c Identi ty

MorphologyPluripotency

SterilityMycoplasmaMorphology

Vector clearance Chromosomal stability Viability


05 Poster_V04.indd 1 09.06.16 21:17

EBiSC: Quality Control


All cell lines supplied from EBiSC are thoroughly characterised in order to sustain a collecti on of high quality iPSC lines. Roslin Cell Sciences, the Nati onal Insti tute for Biological Standards and Controls (NIBSC) and EBiSC depositor centres collaborate closely to provide a comprehensive data set for each cell line.

Geneti c Identi ty

Ladd

er

Posi+

ve Con

trol

Nega+

ve Con

trol

No Template Co

ntrol

Test Sam

ple 1

Test Sam

ple2

Adventi ti ous contaminati on of cell lines can seri-ously aff ect cell identi ty, proliferati on, morpho-logy and behaviour. All lines processed within EBiSC undergo conti nuous monitoring for the presence of microbiological agents such as Mycoplasma and bacteria.

EBiSC will assess and record the geneti c identi ty of each iPSC line to detect and prevent the spread of any cross contamina-ti on of cell lines. This will be done by recording a geneti c fi n-gerprint for each line by assessment of Short Tandem Repeats (STRs). STR loci are varied numbers of sequence repeats found in highly polymorphic stretches of DNA. They are detected by amplifi cati on of a number of these loci and sub-sequent high resoluti on, size-based separati on of amplicons.

Mycoplasma testi ng is performed on in-process samples using endpoint PCR and cell banks for distributi on are screened using the more sensiti ve QPCR method.

All lines also undergo broth inoculati on and conti nuous visual assessments to detect non-specifi c micro-biological growth.

Cell PhenotypeHuman pluripotent stem cells have a well characte-rised phenotype associated with the maintenance of self-renewal. A number of factors contribute to maintaining this phenotype including opti misati on of cell culture conditi ons.

Within EBiSC, cell line phenotype is assessed by quanti fying the expression of self- renewal mar-kers SSEA4, TRA-1-60, OCT3/4 and a lack of SSEA1 expression using fl ow cytometry. Morphology and confl uency checks are also carried out on a daily basis by visual inspecti on to assess colonies for ti ght, defi ned borders and smooth surfaces.

Next Generati onAssessing colony morphology and confl uency by eye is ti me-intensive and, even when performed by an extensively trained operator, is subject to human bias giving variable results. EBiSC will seek to adopt new technologies such as automated imaging systems which will allow far greater standardisati on and higher throughput.

Next Generati onEBiSC will evaluate and adopt new technologies such as KaryoLite BoBs which off ers increased throughput over G-Banding and is more cost eff ecti ve.

Next Generati onEBiSC is acti vely evaluati ng alternati ve technologies to assess pluripotent potenti al, for example, the use of microfl uidic array plates.

Pluripotent Potenti alPluripotency is defi ned as the ability for a cell line to form all post-embryonic lineages. This is determined within EBiSC by spontaneous diff erenti ati on of iPSC lines to early germ layer populati ons. Quanti tati ve PCR is used to assess the expression levels of early germ layer markers, with up-regulati on indicati ng functi onal pluripotency.

Pictures of EBS and QPCR data Stem Cell Markers

OCT4SOX2

Nanog

0.0

0.5

1.0

1.5D0D7D14

Rel

ativ

e ge

ne e

xpre

ssio

n

Day 0 Day 7 Day 14

Chromosomal StabilityChromosomal abnormaliti es such as deleti ons or inser-ti ons of geneti c material may aff ect cell identi ty, proli-ferati on and response to sti muli.

Within EBiSC, karyology by G-banding is used to visua-lise chromosomal structure and to detect genomic ab-normaliti es such as inversions, deleti ons, duplicati ons and translocati ons.

Sterility

As part of the EBiSC Quality Control regime a variety of cell line related data is also recorded or generated where required. This can include:

• Donor informati on and disease background• Nomenclature and identi ti es for traceability

• Cell processing protocols and reprogramming methodology• Confi rmati on of geneti c abnormaliti es


06 Poster_V04.indd 1 09.06.16 21:20

EBiSC: Cryopreservati on


Central EBiSC Operati onsCreate banks of ~ 50 vials1-2 million cells per vial

hESCreg nameEBiSC internal name

Depositor’s Cell Line ID2D Barcode encoding vial

Specifi c BioSamples ID

Vial specifi c BioSamples ID

Final Storage and Distributi on at ECACC

Item TRACKER Soft ware

• Sample management soft ware that allows reliable tracking of vial locati on

Shipping of vials• Use of validated dry shippers• Cooling to -150 maintained for

at least 10 days• Data logger included to monitor main-

tenance of correct temperature• Wide neck ensures whole boxes can be

loaded without the need to manually pick vials into diff erent containers

Automated Cryopreservati on at EBiSC Mirror Bank – Fraunhofer IBMT

Retrieve vials• 2 vials kept at Roslin Cell

Sciences as back up• The bulk is split between

two fi nal cryopreservati on faciliti es

90 %

10 %


07 Poster_V03.indd 1 09.06.16 21:24

Cardiovascular

Liver

Musculoskeletal

Immune system

Kidney

CNS

Testes

GI

Eye

Gene<c Tox

Reproduc<ve Tox

EBiSC: Uti lisati on of iPSCs in Drug Discovery and Development


Uti lisati on of iPSCs in R&D toxicity assays Dose response of cisapride in iPS cardiomyocytes

• in vitro assay• Predicti ve of risk in man• Capable of driving SAR• Short loop ti me• Low cost• Avoid animal studies

Att riti on by target organ toxicity

Uti lisati on of iPSCs in R&D effi cacy assays

Cycle of drug manufacture iPSC uti lisati on Aspirati onal goal for iPSC in R&D

iPS cardiomyocytes taken as an example

Cardiovascular

Liver

Musculoskeletal

Immune system

Kidney

CNS

Testes

GI

Eye

Gene<c Tox

Reproduc<ve Tox

Valves

Vasculature

Myocardium

Electrophysiology

Hemodynamics

0 10 20 30 40 50

Harris et al., (2013) Toxicological Sciences 134(2), 412–426Carlson et al., (2013)

J. of Biomolecular Screening 181203-1211

Make

DesignTest

Drug to market

Target Identi fi cati on

HitIdenti fi cati on Hits to Leads

LeadOpti misati on

CandidatePreclinicalEvaluati on

Clinical Proof-of-Concept

Evaluati on

0

5

10

15

20

25

30

3 10 100 1000

FPDc

(%

cha

nge

from

bas

elin

e)

[Cisapride] nM

DMSO (n=4)Cisapride (n=3)

Arrh

ythm

ias

*


09 Poster_V06.indd 1 09.06.16 21:27

EBiSC: Supply chain


Starti ng material

People Donors Identi fi ed Stored Tissue iPSCs Banked iPSCs Diff erenti ated cells Screening & testi ng resources

Data

Stem cells Stem cells for distributi on Cells for research Research The customer

General public

Potenti al donors = individuals with scienti fi cally relevant ti ssue and willingness to donate ti ssue for research

Medical research chariti es

Disease researchAcademic

insti tuti ons

Commercial companies

Toxicology testi ng

iPSC Centres

Clinicians treati ng pati ents

Researchers interested in

specifi c disease areas

Publicly funded research

networks and resources

Pati ent groups Biobanks Other iPSCs banks

Academic insti tuti ons

Academics

Commercial companies

Diff erenti ated cells are used by academic researchers to investi gate disease aeti ology and to screen for new drugs. Commercial companies use them to screen drugs on relevant cell types and test the toxicity of drugs, chemicals and cosmeti cs.

10 Biobanks of stored ti ssue may be valuable but original donor consent documents must be carefully scruti -nized.

5 Existi ng links between clinics and pati ents are valuable but re-consenti ng for iPSC generati on may be required.

Pati ents groups and specialist medical research chariti es provide access to highly moti vated donors for whom the research into their disease will directly benefi t their families.

1

2

Clinicians and researchers are of-ten cauti ous before allowing subjects to donate ti ssue for commercial or competi ng academic research.

Many territories have established links between researchers and do-nors willing to parti cipate in research and make their medical records avai-lable (eg. Generati on Scotland).

3

4

Many academic and commercial centres are making iPSCs. iPSC sam-ples can be deposited by any centre under a Material Deposit Agreement (MDA).

EBiSC will exchange iPSC lines with other Internati onal iPSC banks.

6

7

Samples of iPSC lines will be dis-tributed to academic & commercial users under a standard Access and Use Agreement (AUA).

iPSCs from EBiSC will be diff eren-ti ated into a variety of cell lineages by academic and commercial resear-chers for use by themselves or others in research.

8

9

2

3

3

7

6

8

8

Drug discovery

Academics

Pharma , & biotech companies

Pharma , & biotech companies

Commercial: in-house

researchers at Pharma & other

drug development companies

10

Academic insti tuti ons

10

Commercial: contract research

organisati ons10


10 Poster_V03.indd 1 09.06.16 21:31

The EBiSC Catalogue


EBiSC launched its online catalogue of induced pluripotent stem cells (iPSCs) on 23 March 2016. The iPSC lines deposited by EBiSC project partners as well as leading external iPSC labs are available to academic and commercial scientists for use in disease modelling and other forms of pre-clinical research. The growing catalogue is being added to in three phases which shall in future cover a large number of disease representative and control lines.

Parkinson’s

SNCA Triplicati onSNCA (A53T)GBA (N370S)GBA (L444P)Lrrk2 G2019SSporadic PD

TARDBP C9ORF72ProgranulinTau splicing mut.

FTD & ALS

Hunti ngton’s

Machado-Joseph

Schizophrenia

Auti sm

Migraine

Epilepsy

Bipolar

Alzheimer’s

ApoE Isogenics (E4/4, 3/3, 2/2, KO)

MAPT (P301S)MAPT (P301L)CD33 (ex2 del)

APP (V717I)TREM-2 (R47H)PSEN1 (M139V)PSEN1 (Y115C)

htt ps://cells.ebisc.org

Phase One (white): The Foundati onal collecti on, available now.

Phase Two (yellow): Existi ng lines being deposited with EBiSC by collaborati ng European projects such as Stem-BANCC, HipSci and the Oxford PD Centre.

Phase Three (orange): New lines being reprogrammed or gene edited through acti viti es funded by the IMI and sup-ported by EFPIA companies.

USERSThe growing catalogue can be viewed online at: cells.ebisc.orgMost lines are available immediately once the purchasing organisati on has agreed to the EBiSC Access & Use Agreement.

DEPOSITOR benefi ts• Single Material Transfer Agreement• Backup storage in case own stocks compromised• EBiSC Quality Assured banking• Data archiving• Maintenance of line ownership• Raised profi le for internati onal collaborati ons

Opti cal

Neuromuscular Neuropathy

Cardiovascular Diabetes

Early T2D Sporadic T2D Monogenic

TRESK

GARS

SCN9aTRPA1

HSP27

CIPAge relatedMacularDegenerati on

FSHD

LQTS1

LQTS2

LQTS3

TNNT2

KCNH2

KCNQ1

CPVT

TNNT13MYH7

MYBPC3BRGDA1

DMD

Reti niti s Pigmentosa

NephrologyHealthyControls

FSGS

01 Poster_V03.indd 1 09.06.16 00:15

Documents

European Bank for induced pluripotent Stem Cells04 Poster_V05.indd 1 09.06.16 00:21 EBiSC: The Banking Process The EBiSC - European Bank for induced pluripotent Stem Cells project