Download ISCT Podigy Cell processing poster

A fully automated cell processing system
for various applications
Iris Spiegel, Elmar Fahrendorff, Markus Granzin, Volker Huppert, Gerd Steffens, and Stefan Miltenyi
Miltenyi Biotec GmbH, Bergisch Gladbach, Germany
3
Introduction
Protocols for the manufacture of cellular products
usually include a series of pre- and post-separation
handling steps. We have developed an integrated cell
processing device, which can handle all current technical
requirements for manufacturing cellular products by
automation of the complete process in a functionally
closed environment.
Microscopic und macroscopic evaluation of PBMCs prepared
with the cell processing device
A
In addition to the camera for layer detection, the cell
processing device includes a microscope camera,
which allows visualization of the cellular products.
Figure 4 shows a suspension of PBMCs prepared by
density gradient centrifugation (A, microscopic view;
B, suspension in the collection bag). Erythrocytes were
reduced to less than 1% (cell volume).
B
Methods
To facilitate important cell manufacturing processes,
such as density gradient centrifugation, cell washing
processes, volume reduction of cell culture suspensions,
cultivation of cells, and red blood cell reduction in a
functionally closed system, we have developed three
different tubing sets. Each tubing set allows several
options for connecting media, buffers, or other
supplements. The tubing sets include a novel, singleuse centrifugation chamber, enabling cell washing and
density gradient–based separations of cell suspensions.
Integrated channels allow liquids to be added or
removed during centrifugation.
Specifically, the tubing sets are designed for:
1. reducing large volumes of cell culture suspensions
2. cell washing processes, density gradient
centrifugation, and red blood cell reduction
3. applications as described under 2) with the addition
of cooling and heating capabilities, as well as gas
control.
We are currently developing user-specific programs,
which – in combination with the tubing sets mentioned
above – allow for adaptation to individual needs.
The novel, single-use centrifugation chamber has been
designed in various configurations to cover a broad
range of applications.
Figure 1
4
Evaluation of granulocyte removal from PBMCs
Figure 5
One of the main goals of PBMC preparation is the
removal of granulocytes. To evaluate the efficiency
of granulocyte elimination by the automated density
gradient centrifugation, we incubated buffy coat and
PBMCs with a CD15-APC antibody for flow cytometric
analysis. Additionally, leukocytes were stained with
We have developed an automated density centrifugation
procedure, which allows separation at a density of
1.077 g/mL. First, the buffy coat sample is transferred
into the centrifugation chamber (fig. 2A). Afterwards,
the density medium is pumped into the chamber
while the chamber is rotating at 400×g (fig. 2B).
During centrifugation the components from
the original sample form layers according to their
different densities. The endpoint of fractionation is
determined by an integrated camera.
The peripheral layer contains erythrocytes and
granulocytes and is followed by density medium layer
and peripheral blood mononuclear cells (PBMCs).
The inner layer includes plasma (fig. 2C).
A
B
C
41 – 77%
96 – 99.2%
5%
0.1 – 4.13%
Figure 6
6
1×10⁹
1×10⁸
1×10⁷
2
0
Figure 7
5
10
Days
15
20
To assess functionality of the PBMCs prepared with
the cell processing device we analyzed, e.g., NK cell
expansion. PBMCs were cultured in the presence of OKT3 and IL-2 for three weeks, resulting in a preferential
expansion of NK cells. All cell culture steps were
executed automatically by the device. CD3–CD56+
NK cells from three different donors were enumerated
by flow cytometry at various time points. The expansion
profiles show a decrease of NK cells during the first few
days, followed by a phase of exponential growth and a
steady state at the end. After three weeks, NK cells were
expanded by 54–156-fold to yield 1.0–5.1×10⁸ cells.
Conclusion
The novel device can perform numerous cell processing
steps, including cell washing and labeling as well as
more complex procedures, such as density gradient
Ray of light passes
through the layers
within the groove
B
Figure 3
We analyzed recovery and viability as well as the
granulocyte content in PBMCs prepared with the
cell processing device. We compared these data to
the technical specifications of the density gradient
medium provided by the manufacturer of the medium.
The recovery ranged between 41 and 77%. Viability
was higher than 96% and the maximum granulocyte
contamination was below 4.5% (n=10). The amount of
erythrocytes was lower than 1% (cell volume). Overall
our results were well within the technical specifications.
Functional analysis of PBMCs
1×10¹⁰
1×10⁵
A
results
> 90%
Maximum
granulocyte content
1×10⁶
Automatic layer detection
Technical
specification
60 ± 20%
Viability
Figure 2
CD45-FITC. The original buffy coat sample contained
21.2% CD15+ granulocytes, whereas in the PBMC fraction
the frequency of granulocytes was reduced to 0.64%.
Dead cells and erythrocytes were excluded from the
analysis.
Recovery, viability, and granulocyte content in PBMCs
Recovery of PBMCs
from the original
sample
NK cell number
Density gradient centrifugation
PBMCs
Buffy coat
5
Results
1
Figure 4
Interphase between
plasma and air
Plasma
To allow for automated separation of the layers,
we developed a proprietary technology for layer
detection, which involves an integrated camera.
During centrifugation, formation of the layers also
occurs within the groove of a double prism, which is
PBMCs Density
medium
erythrocytes +
granulocytes
located inside the centrifugation chamber (fig. 3A).
A light ray passes through the prism’s groove, allowing
the camera to capture images of the layers (fig. 3B). These
images allow the determination of the layers’ volumes,
which are subsequently used for further calculations.
centrifugation. Clean room requirements are markedly
reduced as all steps are carried out in a closed system.