Bioengenharia de Bioengenharia de

células estaminaiscélulas estaminais

Pedro Cruz

Lisboa, Julho 2008

Animal Cell Technology Laboratory

Pancreatic Stem Cells (PSCs)Pancreatic Stem Cells (PSCs)

cells from the exocrine parts of the pancreas (Kruse et al., 2004)

plasticity

Banas et al. (2007) Dev Dyn

interesting candidateinteresting candidatecell pools for tissue regeneration cell pools for tissue regeneration

strategiesstrategies

Pancreatic Stem Cells (PSCs)

plasticity: differentiation into cell types of all three germ layers;

significant self-renewal activity;

excellent viability: easy to cultivate and cryo-preserve.

multiple tissue niches identified as sources for adult stem cells (SCs)

Goal:Goal: development of efficient bioprocesses for development of efficient bioprocesses for large scale expansion of pluripotent PSCslarge scale expansion of pluripotent PSCs

scalable;

fully controlled;

hydrodynamically well-characterized;

culture homogeneity;

reproducible experimental conditions;

easy sampling.

suspension cultures in stirred bioreactorssuspension cultures in stirred bioreactors

RSA Pank: Rat Cell line (PSCs)

without compromising the differentiation potential;

in fully controlled and scaleable culture systems.

Bioreactor technology

Culture StrategiesCulture StrategiesStrategy 2: microcarriers

Day 1

Day 3

125 mL spinner vessels; inoculum: 4x105 cell/mL; 50 - 100 rpm.

• small aggregates (70 – 90 m).

• aggregates increased in size (90-120 m);

Strategy 1: 3D cell aggregates

Day 13

DAPInestin

125 mL spinner vessels; inoculum: 1x105 cell/mL; Cytodex 1 or Cytodex 3: 3g/L (5-8 cells/ microcarrier); 50 - 80 rpm.

no cell proliferation.

spontaneous differentiation.

(aggregate clumping)

Scale bar: 100 m

Cyt1

• cells adhere to both microcarriers and were able to proliferate;

• adherent cells expressed nestin (PSC marker).

Cyt3

nestin

nestin

Scale bar: 100 m

Day 4

microcarriermicrocarrier culture strategyculture strategy suitable suitable for expansion of undifferentiated PSCsfor expansion of undifferentiated PSCs .

0

0.05

0.1

0.15

0.2

0 1 2 3 4 5 6 7 8 9

Days

To

tal

cel

l co

nc

en

tra

tio

n

(x 1

06 cel

ls/m

L)

0.0

1.0

2.0

3.0

4.0

cu

mu

lati

ve

qL

DH

(U/(

106 c

ell

. d

ay

))

Cyt1 [total cell] Cyt3 [total cell] Cyt1 (qLDH) Cyt3 (qLDH)

Microcarrier StrategiesMicrocarrier Strategies

Cyt1 Cyt3

% cells attached to microcarriers ~75% ~90%

fold increase in cell concentration (FI)* 2.2 2.2

growth rate (, day-1) 0.21 0.26

doubling time (Td, day) 3.2 2.6

• PSCs adhered better on Cytodex 3 (collagen layer);

• highest cell concentration achieved with Cytodex 3 (0.19x106 cells/mL);

• faster cell growth on Cytodex 3;

• significant drop on culture viability, with both microcarrier supports, after day 4.

* Considering the % of cells that attached to the supports

Cytodex 3Cytodex 3 microcarriers supported PSCs expansion more efficiently microcarriers supported PSCs expansion more efficiently

PSC Expansion in BioreactorsPSC Expansion in Bioreactors

improveimprove and up scaleup scale PSC expansion:

Next Step …

Cultivation of PSCs on Cytodex 3 microcarriers, in suspension bioreactors

Controlled Environment 37ºC37ºC

pH 7.2pH 7.2

50-80 rpm50-80 rpm

pOpO22: 30% : 30%

Cell expansion and viability

Metabolic performance

Self-renewal capacity

Differentiation potential

Daily monitoring

250 mL vessel

IN THE BIOREACTOR:

• PSCs adhered to Cyt3;

• adherent cells nestin positive (PSC marker);

• from day 5 onwards, cells detached from the microcarriers.

PSC expansion on PSC expansion on Cyt3Cyt3 microcarriers feasible in microcarriers feasible in suspension bioreactorssuspension bioreactors

PSC Expansion in BioreactorsPSC Expansion in Bioreactors

Day 0

Day 9

Day 4nestin

Scale bar: 100 m

PSC Expansion in BioreactorsPSC Expansion in Bioreactors

SP BR

fold increase in cell concentration (FI)* 2.2 4.7

growth rate (, days-1) 0.26 0.35

doubling time (Td, days) 2.6 2.0

in the bioreactor:

• 2-times higher FI

• higher growth rate;

• faster doubling time.

* Considering the % of cells that attached to the supports

SP BR

qGLC, mol/(106cells.day)

13 13

qLAC, mol/(106cells.day)

29 20

YLac/GLC 2.1 1.5

improved cell growthimproved cell growth

YLAC/GLC <2 in the bioreactor:

higher oxygen availability;

more efficient usage of GLC.

higher cell proliferationhigher cell proliferation

PSC Expansion in BioreactorsPSC Expansion in BioreactorsCharacterization of expanded PSCs

Self renewal capacity

Telomerase activity

PSCs expanded in the PSCs expanded in the BRBR presented presented high telomerase activityhigh telomerase activity

essential for extended

replication activity

indication of cell

proliferation potential

Differentiation potential Adipogenic differentiation

PSCs expanded in the PSCs expanded in the BRBR

were able to were able to differentiate differentiate

into adipocytesinto adipocytescytoplasmic lipid droplets stained

with Oil red

ConclusionsConclusions The expansion of undifferentiated PSCs was feasible using The expansion of undifferentiated PSCs was feasible using

microcarriers;microcarriers;

Cytodex 3 microcarriers provided better substrate for PSC Cytodex 3 microcarriers provided better substrate for PSC

adherence and growth;adherence and growth;

The potential of bioreactor technology combined with the The potential of bioreactor technology combined with the

efficient Cytodex 3 strategy greatly improved PSC proliferation;efficient Cytodex 3 strategy greatly improved PSC proliferation;

Fully controlledFully controlled stirred BIOREACTORstirred BIOREACTOR were successfully were successfully

implemented for implemented for expansion expansion ofof pluripotent PSCs pluripotent PSCs..

The BIOREACTOR strategyThe BIOREACTOR strategy developed herein can be applied developed herein can be applied

to other stem cell culturesto other stem cell cultures

Funding: European Commission (CellPROM: NMP4-CT-2004-500039 )FCT (BD and BPD fellowships)

ITQB-UNL/IBET: Animal Cell Technology Laboratory

Paula AlvesManuel Carrondo

Margarida SerraSofia LeiteMarcos Sousa

Hagen von BriesenErwin Gorjup

Fraunhofer - IBMT

AcknowledgmentsAcknowledgments