Stem cell therapies are rapidly progressing from early research through clinical trials. Most, if not all, clinical batches to date have been produced in 2D static culture. Various suppliers are providing an assortment of 2D culture vessels with significantly more surface area to meet these needs. However, 2D culture has significant economic and regulatory drawbacks compared to 3D culture. From a production and scalability perspective it would be ideal to adapt these cell therapy products to suspension growth. Consequently, an easier and quite achievable scale-up method is to culture these anchorage dependent cells on microcarriers suspended in fully-controlled bioreactor.

Culture of stem cells on microcarriers can pose a challenge for traditional stirred tank bioreactors; these cells are often more sensitive to shear forces in the bioreactor than those traditionally culture in suspension. Different adaptations and specialized impellers have been developed over time to facilitate microcarrier culture. However, difficulties remain in suspending particles uniformly with horizontally-rotating impellers without employing fast agitation speed which can induce more shear stress forces to the cells.

The Air-Wheel® bioreactor system from PBS Biotech is designed to overcome this limitation by using a large, vertically-rotating mixing element that provides high particle suspension power with gentle mixing at low power inputs. In the present work, the expansion and migration of hMSC in 3L Air-Wheel® system under xeno-free conditions is described.

Margarida Serra1,2, Marta Silva1,2, Marcos Sousa1,2, João Sá1,2, Manuel Carrondo2,3, Paula Alves1,2

Yas Hashimura4, Daniel Giroux4, Brian Lee4

1 iBET- Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal; 2 Instituto de Tecnologia Quimica e Biologica – Universidade Nova de Lisboa, Oeiras, Portugal; 3 Faculdade de Ciências e

Tecnologia, Universidade Nova de Lisboa, Monte da Caparica, Portugal; 4 PBS Biotech Inc., Camarillo, California, USA

Xeno-free Cultivation of hMSC in Air-Wheel® Bioreactor BM-hMSC (STEMCELL Technologies) were expanded in MesenCultTM-XF (STEMCELL Technologies) medium and passaged using TrypLE Select (Life Technologies). On passage 3,

hMSC were used to inoculate a 3L Air-Wheel® bioreactor (PBS Biotech) at 2.2L working volume, and a control culture was carried out in stirred tank (ST) bioreactor (Sartorius

Biostat Qplus) at 0.25L working volume. The cell attachment to microcarriers was monitored in the first 5 hours and the cell growth profile was monitored daily (Expansion

Phase). On Day 6 of culture, empty microcarriers were added to each bioreactor to promote the migration/growth of hMSC (bead-to-bead transfer), according to the protocol

previously optimized by us (Migration Phase). In both bioreactors, dissolved oxygen was controlled at 20% of air saturation and pH at 7.2. Temperature was set to 37º C.

Intermittent agitation profiles were used during cell inoculation and migration, otherwise kept constant at 15-17 rpm and 40-50 rpm in 3L Air-Wheel® and ST, respectively. Data

acquisition and process control were performed using VxWorks OS (PBS Biotech™, USA) and MFCS/Win (Sartorius) for PBS and Sartorius bioreactors, respectively.

1) The vertically oriented Air-Wheel® mixer along a horizontal axle creates tangential fluid motion with high sweeping power to suspend particles well in the round-bottom vessel

2) The oppositely-oriented internal vanes of the mixer guide fluid bi-axially as it rotates

3) The large size of the Air-Wheel® mixer and the vanes dissipate the energy to the liquid over a large surface area

The combination of these 3 elements offers gentle and uniform fluid mixing and efficient particle suspension inside the vessel even at low power input and agitation speeds.

Day 1 Day 6

PBS

ST

Day 3

Day 7 Day 14

PBS

ST

Day 10

hMSC successfully attached and grew on microcarriers in both PBS and ST bioreactors

hMSC were able to migrate from colonized microcarrier onto empty beads and remained viable through culture time in both PBS and ST bioreactors

The expansion of hMSC on microcarriers was successfully established in a 3L Air-Wheel® bioreactor system under xeno-free culture conditions:

- High cell volumetric productivities (3×105 cell/mL) and enhanced cell migration capacity (as bead-to-bead cell transfer) was observed in PBS bioreactor;

- Higher percentage of proliferating cells was obtained in PBS bioreactor; - hMSC expanded in PBS bioreactor maintained their identity and differentiation potential.

As future work, the combination of expansion and migration steps will be optimized in 3L Air-Wheel® bioreactor system for further improvement in cell volumetric productivities.

hMSC Metabolism

hMSC Characterization & Quality Control Tests

hMSC Viability & Microcarrier Colonization

Proliferation Capacity

Actin Organization of hMSC Cultured in PBS (Day 6)

F- Actin Nuclei (DAPI)

hMSC expanded in PBS bioreactor:

Retained the proliferation capacity over culture time (higher percentage of proliferating cells were observed in hMSC cultured in PBS than in ST bioreactors)

Maintained their immunophenotype

Showed the capacity to differentiate into adipocytes, osteocytes and chondrocytes

Showed a mixed actin organization (actin fibers and cortical actin), related to the efficient cell attachment and growth on microcarriers

hMSC showed similar specific rates of glucose and glutamine consumption and lactate production in both PBS and ST bioreactors

Higher specific rates of glucose and glutamine consumption and lactate production were observed during cell expansion step

Culture Characterization hMSC viability and microcarrier colonization were characterized by fluorescence microscopy using FDA (fluorescein diacetate; green; viable cells) and PI (propidium iodide; red;

dead cells) dyes. Cellular growth was determined using nuclei counting and metabolism was assessed using YSI 7100 MBS system (YSI Lifescience). For hMSC characterization,

the proliferation capacity, cell identity, differentiation potential and actin protein organization were assessed during culture time.

Air-Wheel® Mixing Technology

The authors acknowledge Stephen Caracci and Anne Kwei from Corning Incorporated (USA) for their support in providing Synthemax II microcarriers.

PBS ST Time (Day) 0-6 6-14 0-6 6-14

XInitial (x105 cell/mL) 0.25 2.04 0.25 1.69

XMax (x105 cell/mL) 2.04 3.06 1.69 2.79

mMax (h-1) 0.012 0.011

Expansion Ratio (XMax/XInoc) 12.2 11.0

Population Doubling (PD) 3.6 3.5

Expansion Migration0.0

0.5

1.0

1.5

qG

LC

(mm

ol/10

6cell.h

)

Expansion Migration0.0

0.5

1.0

1.5

qL

AC

(mm

ol/10

6cell.h

)

hMSC cultured in PBS bioreactor showed similar growth profile, expansion ratio and number of population doublings to the hMSC expanded in ST bioreactor

After the addition of empty microcarriers, cell migration was more efficient in PBS bioreactor where a faster microcarrier colonization was observed

hMSC Growth Profiles

0 2 4 6 8 10 12 140

1

2

3

4Addition of

empty MC

Time (day)

Cell C

on

cen

trati

on

(x10

5cell/m

L)

0 2 4 6 8 10 12 140

20

40

60

80

100

Addition of

empty MC

Time (day)

Mic

rocarr

ier

Co

lon

izatio

n (

%)

0 2 4 6 8 10 12 140

20

40

60

80

100

Addition of

empty MC PBS ST

Time (day)

Mic

rocarr

ier

Co

lon

izatio

n (%

)

Expansion Phase

Expansion Phase

Expansion Phase

Migration Phase

Migration Phase

Migration Phase

Percentage of hMSC in Phase S of Cell Cycle (Click-iT EdU Flow Cytometry Assay Kit)

Adipogenic Differentiation Chondrogenic Differentiation Osteogenic Differentiation

(scale bars: 100 µm)

PBS

ST

Differentiation Potential

Cell Identity CD90 CD73 CD105 CD166 CD44 CD45 CD34 HLA-DR

0

20

40

60

80

100

Day 0 Day 14 PBS Day 14 ST

% o

f P

osit

ive C

ell

s

CD90 CD73 CD105 CD166 CD44 CD45 CD34 HLA-DR0

20

40

60

80

100

Day 0 Day 14 PBS Day 14 ST

% o

f P

osit

ive C

ells

Flow Cytometry

Evaluate PBS Bioreactor Performance on hMSC

expansion and migration on synthetic microcarriers

Expansion Migration0.0

0.5

1.0

1.5

STPBS

qG

LC

(mm

ol/10

6cell.h

)

50 µm