Castilho Leda R., Moraes Angela Maria (Ed.) Animal Cell Technology: From Biopharmaceuticals to Gene Therapy
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272 Animal Cell Technology
Figure 2.4
Different morphology types observed in distinct cell lines. (A) Drosophila
melanogaster cell line S2; (B) Spodoptera frugiperda cell line Sf9; (C) adherent
CHO.K1 cell line; (D) CHO.K1 cel l line adapted to su spension growth; (E)
hybridoma producing anti-TNP (trinitro phenol) monoclonal antibody. Bars in
photos (A) and (B) represent 50 m. Photographs (C) and (D) were kindly
provided by Rodrigo Coelho Ventura Pinto (Cell Culture Engineering Laboratory,
COPPE, Federal University of Rio de Janeiro, Brazil), and photograph (E) was kindly
provided by Rita de Ca
´
ssia Paro Alli (Industrial Biotechnology Laboratory, IPT, Sa
˜
o
Paulo, Brazil).
Color Plates
Figure 7.3
Morphological changes in apoptosis and necrosis. (i) A viable nonapoptotic cell
(A), in case of death by necrosis, undergoes a cell swelling process, followed by
cell lysis and release of intracellular material (B). In case of apoptosis, chromatin
condensation near the nuclear membrane (C) occurs and apoptotic bodies (D) are
formed. A final stage, where the cell presents no chromatin (E), may also be
observed. Under in vitro culture conditions, a phenomenon known as secondary
necrosis occurs in the final stages of apoptosis. (ii) Fluorescence microscopy using
acridine orange and ethidium bromide allows identification of the different stages
of cell death: viable nonapoptotic cells (A), necrotic cells (B), viable apoptotic cells
(C), nonviable apopto tic cells (D), and chromatin-free cells (E).
Animal Cell Technology
Figure 9.4
Internal surface of a 1000 L bioreactor for animal cell cultivation. It is also possible
to observe the three-blade impeller and the baffles, which are flat v ertical parts
attached to the inter nal bioreactor surface with the aim of avoiding vort ex
formation.
(A)
(B)
Gas bubbling
Figure 9.5
Stirred-tank bioreactor with a bubble aeration system: (A) schematic
representation; (B) photograph of a 300 L bioreactor (CIM, Cuba).
Color Plates
(B)(A)
Figure 9.6
Wave bioreactor: (A) schematic representation; (B) photograph of a 500 L
bioreactor during a conference exhibition of the company Wave Biotech.
Figure 9.8
Photograph taken under an optical microscope of Vero cells growing on
microporous microcarriers.
(B)
Pump
Gas inlet
Exhaust gas
outlet
(A)
Figure 9.9
Fixed-bed bioreactor: (A) schematic representation including the external
recirculation loop with an aeration tank; (B) photograph of a small-scale
bioreactor.
Animal Cell Technology
(A) (B)
Figure 9.10
Fluidized-bed bioreactor: (A) photograph of a bioreactor commercialized b y G E
Healthcare; (B) operational scheme.
1202.004
R1
SSC-complexity/granularity
10
0
10
0
10
0
10
1
10
1
10
1
10
2
10
2
10
2
10
3
10
3
10
3
10
4
10
4
10
4
FSC-volume
1202.004
M1
FL1-eGFP intensity
Counts
0 40 80 120160200
Histogram Statistics
File: 1202.004
Gate: G1
Total Events: 10000
Acquisition Date: 02-Dec-04
Gated Events: 9800
Marker
All
M1
Left,
1,
17,
Right
9910
8058
Events
9800
8662
% Gated
100.00
88.39
% Total
98.00
86.62
Mean
106.57
119.70
CV
99.56
88.60
Median
78.44
88.17
Figure 10.4
Example of insect cell analysis that expresses the GFP (green fluorescent protein), through a FACS
machine. (A) The abscissa indicates the front dispersion (frontal scatter count, or FSC), that evaluates
thecellvolume(size),whiletheordinateshowsthelaterallightdispersionofeachcell(sidescatter
count, or SSC), that estimates the intracellular granularity (particles and intracellular organelles). As
each cell is positioned in part of the dispersion, it is possible to quantify their subpopulations. (B) The
histogram shows the distribution of the number of cells for each dispersed light intensity. If a limit is
defined, population fractions are established (reprinted by permission from Mariza A. Gerdulo dos
Santos).
Color Plates
Figure 12.7
Optical microscope photo of Streamline-rPrA
1
resin, showing the adsorbent
particles composed of quartz and cross-linked agarose in different proportions.
t (min)
mAU
62.15
64.56
67.51
70.77
73.99
77.36
80.79
Figure 13.4
Capillary electrophoresis of purified rhEPO (recombinant human erythropoietin)
protein. From left to right: isoform nos 2, 3, 4, 5, 6, 7, and 8 (according to
European Pharmacopea).
Animal Cell Technology
(A)
(B)
Auto-scaled chromatogram
Auto-scaled chromatogram
Minutes
Minutes
0.00
0.00
20.00
20.00
40.00
40.00
60.00
60.00
80.00
80.00
100.00
100.00
120.00
120.00
140.00
140.00
⫺0.10
⫺0.10
⫺0.05
⫺0.05
0.00
0.00
0.05
0.05
0.10
0.10
0.15
0.15
0.20
0.20
0.25
0.25
0.30
0.30
0.35
0.35
0.40
0.40
AU
AU
Figure 13.1
Peptide mapping. (A) Peptide mapping of the international standard of rhEPO
(recombinant human erythropoietin) (BRP – European Pharmacopea). (B) Peptide
mapping of an rhEPO production lot.
Color Plates
123
4
5
6
7
Figure 13.2
SDS-PAGE of purified rhEPO (recombinant human erythropoietin). Lanes 1–5:
dilutions 1: 5 o f product (25, 5, 1, 0.2, and 0.04 g, respectively); lane 7:
international standard (25 g).
123
4
5
6
7
8
Figure 13.3
Isoelectric focusing of different samples of rhEPO (recombinant human
erythropoietin).The different profiles of isoforms are observed in the different
preparations. Lane 3: international standard.
Animal Cell Technology
11
Animal cell separation
Leda dos Reis Castilho and Ricardo de Andrade Medronho
11.1 Introduction
After cell cultivation, a cell separation step is generally needed to process
the culture medium to purify the product. This step is performed either to
obtain a cell-free medium for an extracellular product or to obtain a cell
concentrate if the product is intracellular. In animal cell cultures, the
product is normally secreted. Thus, after cell separation, the product is in
solution and can be processed by chromatographic techniques or other
protein purification methods, as presented in Chapter 12.
When the culture is operated in perfusion mode (Chapter 9), the
retention device may be placed inside or outside the bioreactor (Figure
11.1). When the cell culture is operated in batch or fed-batch mode, the
cell separation is conducted after the end of the cultivation period (Figure
11.2). This is the first step of downstream processing.
Animal cell separation is far from a trivial task for many reasons.
Animal cells have no cell wall. They are protected only by the cellular
membrane (lipid bilayer). For this reason, they present high sensitivity to
shear stress. When cultivated in suspension, the cells tend to attach to the
device walls, which may generate clogging problems. Their density is not
much higher than water (1.06–1.14 g cm
–3
) and cell size is in the range of
8–40 m, which results in low settling velocities (Medronho, 2003). For
example, Luebberstedt (2000) worked with HeLa cells with a density of
1.06 g cm
–3
and varying from 10 to 28 m in size. Schultz (1996) found
BHK-21 cell sizes in the 11–25 m range.
Spent medium
Perfusion with
internal device
Fresh
medium
Cell retention
device
Perfusion with
external device
Fresh
medium
Spent medium
Cell retention
device
Figure 11.1
External (left) and internal (right) cell separation device in a perfusion culture.