EFFECTIVE HYPOTHERMIC STORAGE OF HUMAN PLURIPOTENT STEM CELL-DERIVED CARDIOMYOCYTES COMPATIBLE WITH GLOBAL DISTRIBUTION OF CELLS FOR CLINICAL

APPLICATIONS AND TOXICOLOGY TESTING Cláudia Correia1,2, Alexey Koshkin1,2, Madalena Carido1,2, Nuno Espinha1,2, Pedro Lima3, Margarida Serra1,2, Paula M Alves1,2

1iBET, Oeiras, Portugal; 2ITQB-UNL, Oeiras, Portugal; 3Nova Medical School, Lisboa, Portugal

AIM ESTABLISHMENT OF EFFECTIVE CLINICALLY COMPATIBLE

STRATEGIES FOR COLD (4ᴼ C) STORAGE OF hPSC-CMS AS 2D MONOLAYERS AND 3D AGGREGATES

The production of cardiomyocytes (CMs) from human pluripotent stem cells (hPSC) holds great promise for patient-specific cardiotoxicity drug testing, disease modeling and cardiac regeneration [1]. The applicability of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs) in the clinic and industry is highly dependent on the development of efficient methods for worldwide shipment of these cells. We evaluated the feasibility to cold store monolayers and aggregates of functional CMs obtained from different PSC lines using a fully defined clinical-compatible preservation formulation and investigated the time frame that hPSC-CMs could be subjected to hypothermic storage [2].

STRATEGY BACKGROUND

2D MONOLAYERS 3D AGGREGATES

CELL CHARACTERIZATION

HYPOTHERMIC STORAGE

STORAGE SOLUTIONS

STORAGE TIME

MORPHOLOGY STRUCTURE ULTRASTRUCTURE

2D MONOLAYERS

RESULTS

Legend: Nucleus (Nu); myofibrils (MF); Z-bands (Z); neighbouring CMs (CM1, CM2); intercalated disks (ID)

hPSC-CMS WERE STORED FOR 3 (S3), 5 (S5) AND 7 (S7) DAYS AT 4°C IN HYPOTHERMOSOLTM [2]

S7 - Day 1 S7 - Day 7

FD

A / P

I N

UC

VIE

W / M

ITO

VIE

W S7 - Day 1 S7 - Day 7

CELL VIABILITY NECROSIS - FDA (LIVE CELLS) / PI (DEAD CELLS) APOPTOSIS - MITOVIEW (LIVE CELLS)/NUCVIEW (APOPTOTIC CELLS, ACTIVE CASPASE-3)

METABOLIC ACTIVITY RECOVERY PRESTOBLUE ASSAY

HYPOTHERMIC STORAGE OF hPSC-CMS

MF

Z

Z

MF

MF

CM2

CM1

TRANSMISSION ELECTRON MICROSCOPY (TEM)

METABOLOMICS

CULTURE SYSTEM

CELL RECOVERY TRYPAN BLUE ASSAY

3D AGGREGATES

CELL VIABILITY NECROSIS - FDA (LIVE CELLS) / PI (DEAD CELLS)

METABOLIC ACTIVITY RECOVERY PRESTOBLUE ASSAY

SCANNING ELECTRON MICROSCOPY (SEM)

TRANSMISSION ELECTRON MICROSCOPY (TEM)

S7 - Day 7

Legend: Nucleus (Nu); myofibrils (MF); Z-bands (Z); mitochondria (M)

MONOLAYERS OF hPSC-CMS CAN BE EFFICIENTLY COLD STORED FOR 3 DAYS WITHOUT COMPROMISING CELL RECOVERY, METABOLIC ACTIVITY AND ULTRASTRUCTURE

ꭗ CELL VIABILITY DECREASED WHEN THE COLD STORAGE INTERVAL WAS EXTENDED TO 7 DAYS

hPSC-CMS ARE MORE RESISTANT TO PROLONGED HYPOTHERMIC STORAGE-INDUCED CELL INJURY IN 3D AGGREGATES THAN IN 2D MONOLAYERS, SHOWING HIGH CELL RECOVERIES (>70%) AFTER 7 DAYS OF STORAGE

AGGREGATE STRUCTURE/SIZE AND CELL ULTRASTRUCTURE WERE MAINTAINED AFTER 7 DAYS OF STORAGE

1 2 3

0

2 0

4 0

6 0

8 0

1 0 0

S 7 C o n t r o l

B e f o r e S t o r a g e

S I R P A V C A M c T N T

po

si

ti

ve

c

el

ls

(

%)

0 1 2 3 40.0

0.2

0.4

0.6

0.8

1.0

1.2S3 S5 S7

Time post-storage (day)

Fre

qu

en

cy

(in

re

latio

n t

o n

on

-sto

red

ce

lls)

hPSC-CM CHARACTERIZATION FLOW CYTOMETRY

BEATING FREQUENCY

IMMUNOFLUORESCENCE MICROSCOPY

Visit us @ www.ibet.pt Animal Cell Technology Unit, iBET & ITQB-UNL , Portugal Scale-up and Manufacturing of Cell-based Therapies V, January 2017, San Diego, CA, USA

ACKNOWLEDGEMENTS: The authors acknowledge the financial support received from FCT, Portugal (PTDC/BIO/72755/2006), from the European Commission (Hyperlab:223011) and from the FP7 EU project CARE-MI (HEALTH-2009_242038). CC acknowledges the funding from FCT (SFRH/BD/51573/2011).

REFERENCES: [1] Oh, Y., Wei, H., Ma, D., Sun, X. & Liew, R. Clinical applications of patient-specific induced pluripotent stem cells in cardiovascular medicine. Heart 98, 443–9 (2012). [2] Correia, C., Koshkin, A., Carido, M. et al. Effective Hypothermic Storage of Human Pluripotent Stem Cell-Derived

Cardiomyocytes Compatible With Global Distribution of Cells for Clinical Applications and Toxicology Testing. Stem Cells Trans Med. 5;111(3):658-69 (2016).

ELECTROPHYSIOLOGY ANALYSIS

DRUG RESPONSIVENESS

No

rmal

ized

val

ues

N

orm

aliz

ed v

alu

es

CALCIUM TRANSIENTS

AFTER 7 DAYS OF STORAGE, HPSC-CMS MAINTAINED THEIR TYPICAL PROTEIN EXPRESSION PROFILE, SARCOMERIC STRUCTURE, BEATING FREQUENCY, ELECTROPHYSIOLOGICAL PROFILES AND DRUG RESPONSIVENESS

CONCLUSION DEVELOPMENT OF EFFICIENT STRATEGIES FOR

HYPOTHERMIC STORAGE OF 2D MONOLAYERS AND 3D AGGREGATES OF HPSC-CMS

2D MONOLAYER OF HPSC-CMS CAN BE EFFICIENTLY STORED DURING 3 DAYS (80% OF CELL RECOVERY)

3D AGGREGATES OF HPSC-CM PROVIDE BETTER CELL RECOVERY COMPARING TO 2D MONOLAYER AFTER 7 DAYS OF STORAGE

7 DAYS OF HYPOTHERMIC STORAGE DID NOT AFFECT THE PHENOTYPE AND FUNCTION OF HUMAN PSC-CMS STORED EITHER AS MONOLAYERS OR AGGREGATES

STEP FORWARD TOWARDS THE GLOBAL COMMERCIAL DISTRIBUTION OF hPSC-CMS