This led to a recovery of more than 95 % of the cells in the eluate from the column.
The cells were eluted in a wide peak. To evaluate whether the broadness of the peak
was due to heterogeneity among the cells, fractions were collected at the beginning
of the peak and towards the end of the elution. Both fractions showed an identical
elution pattern when re-chromatographed. This means that the peak broadening was
not ascribed to a heterogeneous population of cells, but rather that cells were
somewhat retarded during passage through the column.
However, when applying Escherichia coli cells at low ionic strength, the cells
were captured and then eluted with 0.35–0.40 M NaCl. Recovery of 70–80 % was
obtained. Viability tests showed full viability. The SEM photo in Fig. 6 shows that
the cells were evenly distributed on the gel surface. It is interesting to observe that
the cells are attracted to the plain surfaces and are not held by mechanical entrapment in “dead-flow” zones.
Studies were also carried out using immobilized metal affinity chromatography.
A strain of E. coli with histidine on the surface was passed through iminodiacetic
acid gel (IDA gel). Cells were captured and could not be eluted with high salt
concentrations; up to 1.6 M NaCl was tested. However, imidazol or EDTA released
the cells with a recovery yield of approximately 80 % [55].
Separation of different cells was carried out using an IDA gel. Two different
organisms were used, E. coli and Bacillus halodurans. These two species were
chosen since it is possible to detect one in the present of the other because
B. halodurans grows at pH 10, which is not so for E. coli. Thus, by plate-counting
on plates with different pH values, one could get a clear picture of the distribution
of cells in the different fractions collected after chromatography [55]. The
B. halodurans cells have increased amounts of acidic and hydrophobic amino
acids on the surface, and therefore it was not expected that this cell type would
bind well to the IDA column. The results confirmed this; most of the B. halodurans
cells came in the flow-through peak whereas the E. coli cells were retarded on the
gel and could be eluted afterwards. In all cases, good yields (80 % and upwards) and
viability in the same range were observed.
If cells have relatively similar surface structures, then it may be advantageous to
first specifically modify one group of cells that will be separated from the other. In
the case of lymphocytes, B and T cells have many similarities, but differ in the fact
that T cells have immunoglobulins on their surfaces, whereas B cells excrete
immunoglobulins. By exposing the mixed cell population to a goat antibody
directed against human IgG it was possible to add a separation handle on the B
cells. When passing such a preparation through a cryogel with immobilized
protein A, binding between protein A and the Fc-region of IgG took place and
those cells with goat-anti-human-IgG on their surfaces were retarded, whereas the T
cells passed through without being retarded. After proper washing, the captured
cells were released by a pulse of dog IgG, with a yield of cells in the range of 80 %
and a similar figure for viability (Fig. 7) [56].
The multipoint attachment of cells to the surface of the adsorbent is a real
problem when developing chromatographic separation. After the cells have
attached via a few interactions, more binding takes place that makes it difficult or
even impossible to elute cells in a viable form. During elution, some interactions
Cryogels for Biotechnological Applications
257
The cells were eluted in a wide peak. To evaluate whether the broadness of the peak
was due to heterogeneity among the cells, fractions were collected at the beginning
of the peak and towards the end of the elution. Both fractions showed an identical
elution pattern when re-chromatographed. This means that the peak broadening was
not ascribed to a heterogeneous population of cells, but rather that cells were
somewhat retarded during passage through the column.
However, when applying Escherichia coli cells at low ionic strength, the cells
were captured and then eluted with 0.35–0.40 M NaCl. Recovery of 70–80 % was
obtained. Viability tests showed full viability. The SEM photo in Fig. 6 shows that
the cells were evenly distributed on the gel surface. It is interesting to observe that
the cells are attracted to the plain surfaces and are not held by mechanical entrapment in “dead-flow” zones.
Studies were also carried out using immobilized metal affinity chromatography.
A strain of E. coli with histidine on the surface was passed through iminodiacetic
acid gel (IDA gel). Cells were captured and could not be eluted with high salt
concentrations; up to 1.6 M NaCl was tested. However, imidazol or EDTA released
the cells with a recovery yield of approximately 80 % [55].
Separation of different cells was carried out using an IDA gel. Two different
organisms were used, E. coli and Bacillus halodurans. These two species were
chosen since it is possible to detect one in the present of the other because
B. halodurans grows at pH 10, which is not so for E. coli. Thus, by plate-counting
on plates with different pH values, one could get a clear picture of the distribution
of cells in the different fractions collected after chromatography [55]. The
B. halodurans cells have increased amounts of acidic and hydrophobic amino
acids on the surface, and therefore it was not expected that this cell type would
bind well to the IDA column. The results confirmed this; most of the B. halodurans
cells came in the flow-through peak whereas the E. coli cells were retarded on the
gel and could be eluted afterwards. In all cases, good yields (80 % and upwards) and
viability in the same range were observed.
If cells have relatively similar surface structures, then it may be advantageous to
first specifically modify one group of cells that will be separated from the other. In
the case of lymphocytes, B and T cells have many similarities, but differ in the fact
that T cells have immunoglobulins on their surfaces, whereas B cells excrete
immunoglobulins. By exposing the mixed cell population to a goat antibody
directed against human IgG it was possible to add a separation handle on the B
cells. When passing such a preparation through a cryogel with immobilized
protein A, binding between protein A and the Fc-region of IgG took place and
those cells with goat-anti-human-IgG on their surfaces were retarded, whereas the T
cells passed through without being retarded. After proper washing, the captured
cells were released by a pulse of dog IgG, with a yield of cells in the range of 80 %
and a similar figure for viability (Fig. 7) [56].
The multipoint attachment of cells to the surface of the adsorbent is a real
problem when developing chromatographic separation. After the cells have
attached via a few interactions, more binding takes place that makes it difficult or
even impossible to elute cells in a viable form. During elution, some interactions
Cryogels for Biotechnological Applications
257