2.1. SISTEMAS DE ABASTECIMIENTO DE AGUA POTABLE EN
2.1.2.2. Calidad física, química y microbiológica del agua para consumo
To investigate the influence o f culture system on the growth and IFN-y production of CHO 320 cells microcarrier culture techniques were employed. Nilsson et a l (1988) and Smiley et a l (1989) had both demonstrated the feasibility o f CHO cell production of IFN-y on microcarriers. Three microcarriers were tested for their suitability for CHO 320 cell batch culture: Cytodex 3 (Pharmacia Biotech): a microporous gelatin-coated dextran microcarrier, Cytopore 2 (Pharmacia Biotech) a macroporous cellulose microcarrier and Cytoline 1 (Pharmacia Biotech), a macroporous polyethylene/silica microcarrier. All the microcarriers varied in diameter, relative and charge density and effective surface areas (Section 2.2). Cell yield is directly related to the available surface area and hence the concentration of microcarriers (Nilsson, 1989). Thus in order to provide a better comparison between the first two microcarriers the ratio o f cell density to microcarrier surface area was kept equal by adjusting the microcarrier concentrations accordingly.
The Cytodex culture was set-up at a microcarrier density of 5 g f* and the Cytopore culture at 2 g microcarrier, both with a cell inoculation density of 2 x 1 0 ^ cells ml"\ Growth and detachment o f cells from the microcarrier was observed by fluorescein diacetate staining (Figure 3-43). The growth characteristics on each microcarrier type were similar to those observed in
Chapter 3.6 Investigation of CHO Cell Growth and IFN-v Production in Microcarrier Systems
suspension culture (Figure 3-44a). The Cytopore culture had a typical lag phase and a lower maximum total cell density (0.6 x 10^ cells ml'^) compared to the Cytodex culture. The Cytodex culture demonstrated no lag phase and a typical value for maximum total cell density approaching 1.0 x 10^ cells m l'\ The difference in the extent o f growth and presence o f a lag phase might have been due to the irregular surface morphology o f the Cytopore microcarriers making attachment or cell spreading slower. Alternatively, differences due to the matrix material such as surface charge can influence both attachment and spreading. However, fiirther experiments would be required to determine a definite influence o f microcarrier type.
In addition, it was observed that it was very difficult to remove all the cells from Cytopore microcarriers by the method suggested by the manufacturer. The inclusion o f 1% (v/v) Triton X-100 to the dissociation and staining solution improved the release of nuclei from the microcarriers. However, but some were still observed to have remained within the microcarriers despite extended passaging o f the microcarriers through a syringe needle. The growth rates o f cells on both microcarriers were comparable to batch suspension culture values: the Cytodex culture exhibited a specific growth rate o f 0.019 h'^ compared to a value o f 0.024 h'^ for the Cytopore culture (Table 3-10).
Chapter 3.6 Investigation of CHO Cell Growth and IFN-v Production in Microcarrier Systems
Figure 3-43 A Cytodex 3 microcarrier stained with fluorescein diacetate and observed by fluorescence microscopy. Cells are stained bright green, shadows on surface may represent previous attachment of cells.
t î 1 1 . .
(a)
- • --- O — Cytodex 3 - ■ --- □ — C ytopore 2 I't-B »- B "2=
T im e(h ) Time (h)
Figure 3-44 Growth and production of CHO cells on two different microcarriers, (a) total cell density and (b) IFN-y concentration.
Chapter 3.6 Investigation of CHO Cell Growth and IFN-y Production in Microcarrier Systems
Table 3-10 Growth, productivity and metabolic quotients.
Parameter Cytodex 3 Cytopore 2
(h-') 0.019 0.024
q i F N [ I U (10^ cells)'* h'*] 136 315
qoiucose [mM (10^ cells)'* h'*] 0.246 0.426
qLactate [mM (lO^cells)'* h'*] 0.413 0.608
qAmmonia [mM (lO^CClls)'* h'*] 0.025 0.034
The production of IFN-y was also similar to batch suspension kinetics (Figure 3-44b). Peak IFN-y titres were associated with the period of maximum cell density and remained at the same level during the death phase o f culture. The Cytopore culture exhibited a maximum IFN-y concentration o f 12 x 10^ lU m f’ and the Cytodex culture 10 x 10^ lU ml"\ The mean specific IFN-y production rate (q if n) during the growth phase was 136 lU (10^ cells)'* h'* during the Cytodex culture and 315 lU (10^ cells)'* h * for the Cytopore culture (Table 3-10). The analysis o f metabolites also revealed a very similar series o f profiles when compared to previous batch suspension culture results (Figure 3- 45). Correspondingly, the metabolic rates were also comparable to batch suspension culture data (Section 3.3). However, the metabolite rates o f the Cytopore culture were higher than those o f the Cytodex culture because the total cell densities in the Cytopore culture were lower. Both cultures demonstrated the complete utilisation o f glucose when peak cell densities were reached after about 80 h. Lactate utilisation occurred concurrently reaching a maximum o f ~ 1 6 m M in both cultures at the end o f the culture time.
Chapter 3.6 Investigation of CHO Cell Growth and IFN-v Production in Microcarrier Systems
Ammonia concentrations increased gradually during the two cultures reaching a maximum o f 2 mM. —• — Glucose — ■ — Lactate —A -A m m o n ia P —O — Glucose . — O — Lactate . —A — Ammonia □---
I
I
-- - L A 0 24 48 72 96 120 144 168 24 48 72 96 120 144 168 Time (h) Time (h)Figure 3-45 Metabolite changes during the batch culture o f CHO cells on (a) Cytodex 3 and (b) Cytopore 2 microcarriers.
Every cell culture has a cellular requirement for cell-produced chemicals to avoid long lag phases. Since each microcarrier provides a different microenvironment every microcarrier culture demands a different inoculation density for optimum growth. Since cells do not readily migrate from one bead to another a minimum threshold number of cells per microcarrier is required for inoculation. For example, Hu et a l (1985) demonstrated that the critical number of cells per dextran bead was 6 for human foreskin fibroblasts. Therefore it is common to determine the inoculum concentration o f cells per
Chapter 3.6 Investigation of CHO Cell Growth and IFN-v Production in Microcarrier Systems
microcarrier for successful cultivation. Cytodex 3 was chosen for future study because of its good optical properties and the ease and reliability of cell removal.
Four cultures using Cytodex 3 were set up at different microcarrier densities but with an equal cell inoculation density o f 2 x 10^ cells ml"\ This corresponded to an inoculum number of cells per bead of 50, 25,10 and 1 cell per bead for microcarrier densities o f 1, 2, 5 and 10 g 1'^ respectively. In both the 1 g r* and 2 g 1'^ cultures the microcarriers grew to confluence (Figure 3- 46a). At a microcarrier concentration o f 5 g l '\ although there was more surface area available for growth the extent o f growth was less than for the lower microcarrier density cultures. It was also observed that after one day’s growth some of the microcarriers had none or very few cells attached to them and many remained in this state until the end of the time course. The 10 g 1'^ culture displayed a poor growth curve.
The maximum number o f cells per bead, Xmax(Dm), where x^ax is the maximum cell density at confluence and Dm refers to microcarriers of mean diameter, can give an indication o f the capacity o f each microcarrier for cell growth (Table 3-11). This was calculated from the maximum total cell density at confluence, microcarrier density and the number of microcarriers per g diy weight: 4x10^. This demonstrated that the multiplication ratio o f cells from the inoculation ratio of cells per microcarrier increased by 4.5, 4.3 and 3.5-fold for the 1, 2 and 5 g cultures respectively. These values are typical for batch culture but
Chapter 3.6 Investigation of CHO Cell Growth and IFN-v Production in Microcarrier Systems
could possibly be improved by using microcarriers o f selected diameter and an improved medium (Hu and Wang, 1986). The 10 g 1'^ culture proved too high a density of microcarriers to achieve normal growth and was not analysed further. This suggests that although the minimum inoculation density is between 1 and 10 cells per microcarrier a better cell yield is obtained with at least 25 cells per microcarrier.
Recombinant IFN-y production for the three densities o f microcarrier culture was very similar (Figure 3-46b). However, maximum IFN-y concentrations were higher than those normally encountered for batch suspension cultures by about 2-fold. Most animal cells are most productive when attached to a surface especially if they are o f an epithelial morphology such as ovary cells. At the point of maximum cell density the IFN-y concentration was the same as that achieved during batch suspension culture. However, the IFN-y level continued to rise which was rarely seen during suspension culture. In addition, these levels were not seen during the initial Cytodex 3 experiment (Figure 3- 44). The specific IFN-y production rates for the three cultures during the exponential growth phase were similar to those obtained during suspension culture (Table 3-11). The 1 g 1'^ culture demonstrated a higher rate due to the greater IFN-y concentrations achieved during the initial 48 h (Table 3-11).
Chapter 3.6 Investigation of CHO Cell Growth and IFN-v Production in Microcarrier Systems 1.0-, - ■ - 2 g r ‘ - A - 5 g r ' — ♦ — 10 g I 0.8- 0.6- 0.4- 0.2- 0.0 0.5 - 0.5 0.4 - 0.4 ^ 0.2 H 0.2 A - A A - A A 48 0.0 0.0 0 48 96 144 0 96 144 192 Time (h) Tim e (h)
Figure 3-46 Growth and IFN-y production of CHO cells on Cytodex 3 at four microcarrier densities, (a) Attached total cell density, (b) IFN-y concentration,