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Cryogel applications in microbiology

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Review

Cryogel applications in microbiology

Fatima M. Plieva1, Igor Yu. Galaev2, Wim Noppe3 and Bo Mattiasson2
1
Protista Biotechnology AB, IDEON, SE-22370 Lund, Sweden
2
Department of Biotechnology Lund University, P.O. Box 124, SE-22100 Lund, Sweden
3
Interdisciplinary Research Center, Katholieke Universiteit Leuven Campus Kortrijk, E. Sabbelaan 53, B-8500 Kortrijk, Belgium

There is a great demand for improved technologies considered and re-evaluated. This has highlighted the
with regard to rapid processing of nano- and micro- need for simple technologies to access highly purified
particles. The handling of viruses in addition to phages.
microbial and mammalian cells requires the availability Technologies involving the use of macroporous poly-
of appropriate adsorbents. Recent developments in meric materials which allow for the processing of different
macroporous gels produced at subzero temperatures cell populations are of great interest. Cryogelation (or
(known as cryogels) have demonstrated an efficiency gelation at subzero temperatures) is a recent promising
for processing cell and virus suspensions, cell separ- technology that permits the formation of macroporous
ation and cell culture applications. Their unique com- hydrogels (so-called cryogels) with controlled porosities.
bination of properties such as macroporosity, tissue- One of the most attractive features of cryogels is their
like elasticity and biocompatibility, physical and chemi- macroporosity for processing particulate-containing fluids
cal stability and ease of preparation, renders these including suspensions of microbial cells [4–6].
materials interesting candidates for a broad range of Expanded bed chromatography is a suitable technology
potential applications within microbiological research. for use with crude cell feedstock, however it requires
This review describes current applications of macro- specialized equipment [7,8]. Tangential flow microfiltra-
porous cryogels in microbiology with a brief discussion tion, centrifugation, depth filtration and expanded-bed
of future perspectives. chromatography have been employed for the initial har-
vest of therapeutic products from mammalian cells [9].
Technologies for processing cell suspensions Nevertheless, the trends towards high-density cell cultures
The need for a simple platform when it comes to the and the corresponding augmentation in the level of cell
quantification of cells and the isolation of specific cell lines debris, result in unacceptable fouling of membrane sys-
from various populations has become evident in biological tems, rendering them inefficient. In the early 1990s, mono-
research and diagnostic medicine. The use of immobilized lithic columns (i.e. columns prepared as one piece) started
cell preparations in biotechnological processes allows for a to be used as appropriate separation media for processing
straightforward development of continuous processes with large biomolecules [10,11]. The recently developed, ultra-
an increased productivity, a facilitated product recovery in short, monolithic columns based on cross-linked copoly-
addition to a refinement and re-use of immobilized bioca- mers of glycidyl methacrylate (known as Convective Inter-
talysts. Immobilization is often known to increase the action Media, [CIM] discs) have been extensively studied
operational stability of cells, protect them from the effects for their capabilities of processing viruses [12–15] and
of extreme pH, toxic compounds and also reduce the con- virus-mimicking synthetic particles [16]. However, the
tamination of cell cultures [1]. For mammalian cell culture macroporosity of the reported and commercially available
applications, immobilization allows for an increased cell monolithic adsorbents (up to 3 mm in size) was insufficient
density and results in a simple separation of media from for handling microbial and mammalian cells. The macro-
cells, thereby providing an appropriate cell culture porous cryogels (especially when in the shape of monolithic
environment and an increased productivity. A major issue columns) are currently the only available macroporous
concerns the development of new polymeric supports for chromatography media for processing microbial and mam-
the cultivation of mammalian cells that represent sources malian cells [6].
for a variety of therapeutic products such as antibodies and The use of cryogels in microbiology goes back to the
enzymes. 1980s when they were proposed as matrices for cell immo-
The demand for high-purity, large (at the molecular bilization via mechanical entrapment (see Ref. [17] for a
scale) bioproducts, including viruses and plasmid DNA review). It is the unique properties of macroporous cryogels
as gene therapy vectors, macromolecular assemblies as that provide them with their superior performance with
drug delivery vehicles and virus-like particles as vaccine various cells, compared to other available macroporous
components, has highlighted crucial bottlenecks in down- polymeric matrices commonly used for cell immobilization,
stream processing [2]. Bacteriophages have been recog- such as alginate, carrageenan and polyacrylamide
nized as effective therapeutic agents for antibacterial [1,18,19]. However, the potential of cryogels for cell iso-
therapy as early as the 1940s [3], and the potential of lation and cell culture applications were realized only very
phage therapy for clinical purposes has recently been re- recently [4–6]. Here, we briefly summarize the main appli-
cations of macroporous cryogels in microbiology and offer
Corresponding author: Mattiasson, B. ([Link]@[Link]) some future perspectives.
0966-842X/$ – see front matter ß 2008 Elsevier Ltd. All rights reserved. doi:10.1016/[Link].2008.08.005 Available online 4 October 2008 543
 Author's personal copy

Review Trends in Microbiology Vol.16 No.11

Macroporous cryogels: preparation and properties variety of biological targets. Depending on the potential
During cryogel production under semi-frozen conditions, application, the technological platform of cryotropic gela-
the growing ice crystals perform as porogen. The shape and tion renders it possible to produce materials that are
size of the formed crystals, thus, determine the shape and attractive as potential cell scaffolds [20–22] in addition
size of the pores that develop after defrosting of the sample. to monolithic stationary media for the chromatography of
These highly porous polymeric materials can be produced cells [23–25] with large pores and porosities up to 90%. It is
from essentially any gel-forming precursors and with a also possible to obtain materials with smaller pores and
broad variety of morphologies and porosities [5]. Moreover, thicker pore walls, or with an active filler incorporated
controlling the pore size allows for optimizing the avail- (composite cryogel systems) presenting sufficiently large
ability of the ligands coupled to the polymer backbone for a interfaces to interact with the target biomolecules [26], or

Figure 1. A schematic representation of the two main approaches for cell immobilization. (a) Via mechanical entrapment of cells into cryogels during the preparation of
cryogels of varying format, and (b) through the attachment of cells to the already prepared cryogel systems bearing specific ligands for cell binding.

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even cryogels with small pores of 1–2 mm that can be used ation), the cell suspension is frozen at an optimized freez-
for immobilization of cells through mechanical entrapment ing regime and in a specified geometric format. The latter
[17]. One of the principal differences between cryogels and depends on the properties of the chosen polymeric system
other macroporous materials (with pores of similar sizes) is in addition to the potential applications of the ICBs. Cryo-
that the cryogels possess a tissue-like elasticity and with- gels can be prepared as beads, sheets, monoliths and rods,
stand extensive deformations without being destroyed or can be designed inside an open-ended protective plastic
[27,28]. housing (Figure 1a). ICBs with pore sizes up to 1–2 mm are
produced, in most cases, as beads or sheets [17,29,30]. ICBs
Cryogels for cell immobilization with large interconnected pores of sizes up to 200 mm are
The unique porous structure of cryogels in combination frequently prepared as high-flow path monolithic columns
with their elasticity and high mechanical stability has or discs [31,32] or are produced inside an open-ended
motivated their wide use as matrices for cell immobiliz- protective plastic housing (defined as Macroporous Gel
ation. Depending on the pore size of the cryogels and the Particles, MGPs) to keep the ICBs stable under intense
potential applications of immobilized cell biocatalysts stirring [33,34]. For example, an ICB with a high cell load
(ICBs), two main approaches for cell immobilization are was prepared via mechanical entrapment of yeast cells in
used, namely immobilization via mechanical entrapment the thin pore walls of a dextran-cryogel inside a protective
in cryogels (Figure 1a) and immobilization to the surfaces housing [33] (Figure 2a). It is important to emphasize that
of pores within cryogels via specific interactions or through most cryogel-based ICBs can be stored in a dried state and
adsorption (Figure 1b). be re-activated by swelling before use, with only a minor
Typically, the immobilization of microbial cells via loss of cell viability because of the high stability of the
mechanical entrapment in cryogels is simple. A suspension cryogel matrix [33].
of cells is mixed with monomer (polymer) gel precursors Among the cryogels used for the immobilization of
until a stable cell suspension is achieved. After addition of microbial cells via mechanical entrapment, the cryogels
an initiating system (in case of free radical polymeriz- made from poly(vinyl alcohol) (PVA-cryogels) have proven

Figure 2. Scanning electron micrographs of various ICB preparations. (a) Yeast cells immobilized into pore walls of dextran cryogel and designed inside a protective plastic
core (Reproduced,with permission, from Ref. [33]). (b) E. coli cells attached onto the pore surface of an ion-exchange pAAm cryogel column (Reproduced, with permission,
from Ref. [5]). (c) E. coli cells entrapped in agarose and introduced onto the surfaces of a pAAm cryogel monolith through a ‘double-freezing’ approach. (d) E. coli cells
entrapped in PVA and formed inside the interconnected pores of a pAAm cryogel monolith through the ‘double-freezing’ approach (Reproduced, with permission, from Ref.
[33]). (e) The cultivation of a hybridoma cell line M2139 on a gelatin-coated pAAm cryogel monolith (Reproduced, with permission, from Ref. [34]). (f) Yeast cells on the
surface of an ion-exchange pAAm cryogel monolith.

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to be promising materials, mainly because of the excellent from 6.5% weight/volume PVA, i.e. a concentration much
operational characteristics of PVA-cryogels [17] and bio- lower than those required for preparing mechanically
compatibility [35] and availability of PVA. Both bacterial stable PVA-cryogels) with entrapped E. coli cells was
and yeast cells have been immobilized in PVA-cryogels (see formed inside the interconnected pores of the pAAm cryo-
Ref. [17] for a review article and Refs [36–43] for some gel preformed inside a protective housing. The total con-
recent publications). For example, PVA-cryogel-entrapped struction provided a comfortable environment for the cells,
mycelium of the fungus Rhizopus oryzae was used to and at the same time allowed for an intensive stirring
produce L(+)-lactic acid [36,42]. A well-developed inside the fermentor [33] (Figure 2d). The prepared ICBs
mycelium of Rhizopus oryzae was formed inside ICB beads, were able to withstand long storage times in their dried
confirming a sufficient supply of oxygen and nutrients to state and were found to maintain a high cell activity after
the entrapped fungus [36]. PVA-cryogels were shown to be re-activation in a nutrient medium [33].
very effective for the long-term cryopreservation of Gram- The immobilization of cells into macroporous cryogels
positive and Gram-negative bacteria, yeasts and filamen- with high enough porosities to be used as monolith col-
tous fungi after storage at 188C for 18–24 months [44]. An umns has allowed for the preparation of efficient mono-
excellent stability of the PVA-cryogels permitted the use of lithic bioreactors with enhanced mass transport of
ICBs in a water-poor medium [30,38,39]. For example, substrates and metabolites to and from the entrapped
PVA-cryogel-entrapped cells of Bacillus pseudofirmus cells. Thus, hybridoma cells and human colon cancer
AR-199 with b-galactosidase activity were employed for HCT116 cells could be effectively attached and cultured
the continuous synthesis of alkyl galactosides by transgly- in gelatin-coated pAAm monolith bioreactors for long-term
cosylation, when the synthesis of hexyl galactoside was continuous production of monoclonal antibodies [48] and
combined with a continual product removal [30]. urokinase [49], respectively. The bioreactor for antibody
Long-term use of a single ICB is important for the production was also designed in another format, namely as
development of an efficient industrial process. An import- a reactor filled with macroporous gel particles, MGPs (i.e. a
ant issue is the regeneration of ICB by simple incubation in macroporous cryogel matrix prepared inside an open-
the growth medium to restore the bioconversion activity. ended protective plastic housing) [34] (Figure 2e). Differ-
Another important concern is the preservation of the ICBs entiated mouse 3T3-L1 fibroblasts cultured onto gelatin-
to avoid contamination after prolonged utilization times. coated pAAm MGPs were found to have a high biological
From these perspectives, microorganisms selected from function. This could be demonstrated by a level of released
extreme environments (extremophiles), would constitute glycerol from the differentiated adipocytes almost fivefold
the best choice for the preparation of ICBs. For instance, higher than for control samples (i.e. non-differentiated
PVA-entrapped alkaliphilic Bacillus agaradhaerens, has fibroblasts) [33]. It is believed that MGPs represent novel
demonstrated a high stability during storage in the and promising materials for mammalian cell culture, for
absence of additives. The activity of the stored ICB was which the problems of nutrient transfer and oxygen limita-
completely revived by activation in the culture medium tion are markedly reduced.
[29]. Thus, cells entrapped in PVA-cryogel present an High-density cell suspensions can be applied to mono-
excellent inoculum. No breakage of the cryogel beads lithic cryogel columns without blocking the macropores. In
(because of the cell growth inside the elastic beads) was this manner, yeast cells can be immobilized onto the sur-
observed after at least nine cycles of repeated cell growth face of ion-exchange monolithic cryogel columns bearing
with an enzyme release profile (for cyclodextrin glycosyl- tertiary amino groups by a continuous circulation of dense
transferase) similar to that exhibited by free cells [29]. cell suspension through the column. The attached cells are
Another immobilization strategy includes, as a first uniformly distributed on the pore surface within an ion-
step, the preparation of cryogels with specific ligands used exchange cryogel (Figure 2f). Despite the formation of the
to bind with the cells (Figure 1b). The presence of such multilayer cell aggregates on the surfaces of the cryogel
appropriate ligands on the pore surface within macropor- pores, the back pressure generated by the yeast-loaded
ous cryogels has rendered it possible to specifically bind cryogel column remains low, thus allowing for high flow
viruses [2], microbial [23,24,45,46] and mammalian cells rates of the processing fluid ([Link], unpublished).
[25,47] or separate them from the applied mixture with
other cells. The bound cells were then uniformly distrib- Isolation and separation of cells using cryogels
uted within the entire mesh-work of pores inside the The chromatographic separation of microbial cells is
monolith column without any mechanical entrapment of usually regarded as a very difficult endeavor and many
the cells, as confirmed by scanning electron microscopy factors should be taken into account, including the large
studies (Figure 2b). size of the cells in addition to their low diffusivity, complex
Highly active ICBs have been prepared through a so- surface chemistry and the possibility of multi-point attach-
called ‘double-freezing approach’, via formation of a sec- ment to a surface. In fact, because of the absence of
ondary cryogel network inside the interconnected pores of appropriate sorbents with porosities high enough for pro-
a preformed cryogel [21]. As an example, the double-freez- cessing cells, cell chromatography has thus far not been
ing approach has allowed for an elegant immobilization of routinely used [50]. As a result of the presence of large (10–
Escherichia coli cells by entrapping them in an agarose 100 mm) interconnected pores and mechanically strong
layer within interconnected pores of a polyacrylamide pore walls, the cryogels can withstand high applied flow
(pAAm) cryogel (Figure 2c) ([Link], unpublished). rates with interconnectivity of macropores and practically
In another example, soft and loose PVA-cryogel (prepared no compression, thereby allowing for a convective mass-

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transport of solutes and particles of at least 10 mm in size cells was the physical dislodging of the cells from affinity
[23,51]. When there are no specific ligands on the surfaces surfaces, followed by the removal of the dislodged cells with
of cryogels, the cells freely pass through such monolithic the flow of the squeezed-out eluent. Cryogel monoliths
columns (known as naked columns) without blocking the have high mechanical stability and can, thus, be repeat-
pores. However, when specific ligands are present on the edly compressed without collapsing. Cryogel monolith col-
surface of the pores, the microbial cells can bind to them umns are presently the only available macroporous
and be eluted from the cryogel columns at high yields and adsorbents suitable for chromatography of cells [23].
with a retained viability by means of conventional elution
methods [23,24,45,52]. Cultivation of cells on 3D cryogel scaffolds
In such a manner, pAAm-cryogel columns of various A variety of mammalian cell types have been grown in 3D
functionalities (e.g. ion-exchange, affinity and hydro- cryogel scaffolds, showing an adequate cell response to the
phobic) have been successfully used for the chromato- specific stimulation. The cryogels met most of the require-
graphic separation of microbial [6,23,24,45,52] or ments concerning their suitability as supports for cell culti-
mammalian [25,47] cells. For instance, an almost complete vation. The open-porous structure of 3D cryogel scaffolds
separation of E. coli and yeast cells was achieved under with bioactive surfaces and tissue-like elasticity ensured an
optimized conditions upon application of a mixture of these appropriate microenvironment for the living cells. Various
cells onto a Concavalin A-cryogel (ConA-cryogel) column mammalian cells have been grown in 3D cryogel scaffolds for
[52]. Unbound E. coli cells were detected in flowthrough different purposes [20,33,34,46,48,49,54,55]. The size of the
(Figure 3i and ii), whereas the yeast cells that were affi- pores, the surface chemistry and the pore wall elasticity can
nity-bound to the ConA-cryogel were recovered with a 95% be varied to a large extent when preparing the cryogel
purity when the specific elution (using 0.3 M methyl a-D- scaffolds for cell culture applications. By introducing special
mannopyranoside) was combined with mechanical com- functionalities as extracellular matrix (ECM) recognition
pression of ConA-cryogel monoliths (Figure 3iv) [52]. elements or bioactive substances that induce tissue in-
The high elasticity of the cryogels allowed for the principle growth, it is possible to design the 3D cryogel scaffolds with
difference (compared to the conventional elution methods) bioactive surfaces. The double-freezing approach allows for
of the elution of the affinity-bound cells. In other words, a the broad design of cryogel scaffolds with an open permeable
chemical (or specific) elution accompanied by an extensive structure, an increased mechanical strength and the
mechanical compression of the cryogel monoliths, resulting required gel surface chemistry [21].
in an efficient detachment of the bound cells with a high Various types of cells have been shown to grow and
recovery and a retained viability, was used [27,53]. proliferate on 3D cryogel scaffolds specially designed for
This novel and effective elution strategy, together with a standard 96-well plates. Such a platform could, for
continuous macroporous structure of cryogel monoliths, instance, represent a unique in vitro model for cell-based
renders these adsorbents particularly attractive for appli- assays in drug screening. Consequently, much lower sen-
cations in affinity cell separation. The main strategy for the sitivity of cancer cells to drugs such as cisplatin and Ara-C
separation of mammalian cells involves first labeling the has been demonstrated for cancer cells grown on ECM-
cells of interest with specific antibodies, followed by appli- functionalized cryogel scaffolds in 96-well format, com-
cation to the Protein A-cryogel column and, eventually, the pared to non-functionalized cryogels [46].
release of affinity-bound labeled cells via mechanical com-
pression of the protein A-cryogel monolith. Interestingly, Processing of viruses and phages using cryogels
only affinity-bound cells are eluted in a mechanically Recent developments in cell biology have resulted in new
assisted manner, whereas the release of affinity-bound technologies for the improved detection, purification and
proteins is unaffected by the deformation of the cryogel separation of viral materials. Traditional methods used for
matrix [28]. It can be assumed that the main driving force the selection and purification of viruses such as ultracen-
for the compression-induced detachment of affinity-bound trifugation and filtration are time-consuming, expensive

Figure 3. A chromatogram of a mixture of E. coli and yeast cells on a ConA-cryogel monolith column (112.8 mm long  7.1 mm in diameter). The chromatographic run
comprised the following steps: loading, washing from unbound cells at a flow rate of 21 cm h 1 (i), washing at an elevated flow rate of 430 cm h 1 (ii), conventional elution
with 0.3 M methyl a-D-manno-pyranoside at a flow rate of 430 cm h 1 (iii) and elution by compression of the column bed (iv). Reproduced, with permission, from Ref. [52].

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and very often inefficient. Monolithic chromatography

using CIM discs represents a novel chromatographic
approach for an effective and rapid separation of viruses
[15]. However, these monolithic media do not allow for
processing of non-clarified feeds such as whole-cell broths.
Contrarily, macroporous cryogel monoliths, bearing
specific ligands (e.g. streptavidin) have been used for cap-
turing biotinylated moloney murine leukemia viruses
directly from cell crude feed without clarification [2].
Despite rather low recoveries (less than 10%), the specific
titer in the peak elution fraction was increased 425-fold [2].
Since its introduction by G.P. Smith in 1985 [56], phage
display technology has evolved into a very powerful tool for
the screening of proteins or peptides that bind to a specific
target [56]. The insertion of a foreign DNA fragment in the
genome of a bacteriophage permits the expression of a
fusion protein on one of the phage envelope proteins. In
this way, a library of the different peptides present at the
surface of phage particles is produced. Recently, a new so-
called chromato-panning procedure, based on using a tar-
get molecule that has been covalently immobilized within
the pores of a monolithic cryogel column, has been intro-
duced (Noppe, W. et al., unpublished.). A phage library
with a 6-mer peptide expressed on the pIII protein of
filamentous M-13 bacteriophage was loaded onto the cryo-
gel column with immobilized human lactoferrin. This was
followed by a washing-out of non-bound cells (Figure 4).
Subsequently, a suspension of E. coli cells was loaded onto
the column with bound phages for infection, and finally, the
cells were eluted together with the phages. The amplified
phages were recovered and re-applied to the column for a
second round. A minimal secondary enrichment of selec-
tive clones was observed between the first two rounds with
regard to the affinity towards human lactoferrin. This
indicated that one chromato-panning round was essen-
tially sufficient to obtain high affinity binders–at least in
the case of this system.
The chromato-panning procedure has a few advantages
when compared to a conventional biopanning procedure.
The cryogel column with immobilized target could be pre-
pared in advance, thus omitting the coating step which in
conventional methods could take between a few hours and
an overnight incubation (Box 1, Table I). The cryogel
Figure 4. A schematic representation of a bio-panning procedure with a cryogel
column can be prepared in advance and stored for long monolith column. A 6-mer peptide library expressed on pIII protein of filamentous
durations because of a covalent binding of the target M-13 bacteriophage was used for panning against human lactoferrin. The phage
protein to the matrix. The incubation time is also drasti- library was loaded onto the cryogel column with immobilized lactoferrin and non-
bound phages were washed out (Step 1). Subsequently, one column volume of the
cally reduced, from a few hours or overnight incubation to a E. coli cell suspension was loaded onto a pre-warmed (378C) column with bound
matter of minutes. As traditional procedures usually phages for infection. After incubation, the cells were eluted together with the
require three panning rounds, 50% of the total time phages with 1 M NaCl, and instantly diluted with LB buffer to maintain the viability
of the cells (Step 2).
can be saved. Moreover, the elution step of the phages
from the immobilized target is performed after infection. ture from the cell suspension with its initial purification.
Thus, the risk that strongly bound phages are not eluted No blockage of pores with cells or cell debris inside cryogels
and hence lost, is omitted (Noppe, W. et al., unpublished.). has been observed. For example, Cu(II)-loaded iminodia-
cetae cryogel monolith columns were used for a direct
Capture of soluble products from cell broths capture of extracellularly expressed (His)6-tagged single
Innovative downstream technologies are capable of proces- chain Fv fragments [(His)6-scFv] from whole E. coli cell
sing feeds that contain particulates. Owing to the presence culture broth [57]. It was shown that the purification factor
of large interconnected pores, the macroporous cryogels, remained unchanged upon increasing the applied flow-
bearing specific ligands, can be used for the capture of rates from 50 to 600 cm h 1 [57]. In another example,
specific targets (products) directly from cell suspensions, one of the bacteriocins (hydrophobic peptides that display
thus enabling an integration of the primary product cap- antimicrobial activity against other bacteria) (i.e. sakacin

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Box 1. Fundamentals of the bio-panning procedure

Many variations of the bio-panning procedure are described in the However, the use of basic buffers or specific elution reagents has also
literature, but the different steps of the procedure are principally the been reported [63]. (iv) The eluted phages are incubated with E. coli
same. (i) The target protein is adsorbed to immunotubes, microtiter cells for infection and amplification. Only the infected E. coli cells will
plates or beads. After washing to remove unbound target protein, a survive and grow on the antibiotic-containing agar plates. Microbial
postcoating step is strongly advised to cover the remaining binding replication is an efficient way of producing numerous exact copies of
sites on the matrix and eliminate so-called ‘plastic binders’ that could original phage particles. Not surprisingly, this approach is used to
otherwise interfere with the bio-panning procedure by producing amplify selected phages for the next panning round.
false positive results [62]. (ii) The phage library is incubated with the The biopanning procedure is usually time-consuming and laborious.
adsorbed target protein during a few hours or overnight. The A mean timeframe of the procedures, as listed in Table I, varies
stringency of the next washing step influences the heterogeneity of between 30–65 h for a single panning round, depending on the applied
the eluted later phages. The more stringent the washing, the lower is protocol. Because three and sometimes up to five or six panning
the fraction of weaker binding phages in the remaining population. rounds are necessary to select high affinity ligands, the entire
(iii) Most frequently, an elution with an acidic buffer pH2 is used. procedure requires between one and two weeks to be completed.

Table I. The time frame for chromato-panning as opposed to conventional biopanning procedures
RISEa [59] Target incubation 18 45 h 3 additional identical selection rounds 180 h
Washing-postcoating 4
Cell growth 3
Infection 3
Washing 0.50
Cells-phage incubation
With target 16
Phage recovery 0.50

Microtiter plate Target incubation 2 30 h 2 addition identical 90 h

[60] Washing- postcoating 1.50 selection rounds
Phage incubation 1
Washing 0.25
Elution 2
Infection-amplification 5
Phage recovery 18
Tube panning Target coating 18 62 h 2 additional rounds are 152 h
[61] Washing-postcoating 2.25 performed With phage
Phage incubation 20 incubation of 2.50 h
Washing-elution 0.50
Infection 0.50
Plating-growth 18
Phage recovery 3
Chromato-panning b ROUND 1: (h) TIME per round 23 h No additional panning Total TIME 23 h
Column equilibration 0.25 rounds are needed
On-column panning 1
On-column infection 0.5
Elution 0.25
Plating (amplification) 18
Phage recovery 3
ROUND 1: (h)
a
RISE, Rescue and in Situ Selection and Evaluation.
b
Noppe, W. et al., unpublished.

P) was captured directly from the suspension of the bac- biocompatibility and chemical and mechanical robustness,
teriocin-producing strain of Lactobacillus sakei by using opens up new opportunities for the design of macroporous
cryogel monolith columns bearing hydrophobic functional- materials of superior performance for the processing of
ities [58]. The bacteriocin, captured directly from the dense microbial cells. Mild conditions for the immobilization of
cell broth on phenyl-cryogel columns, was then eluted with cells and a high mechanical stability of the cryogels allow
more than an 80% recovery in the cell-free eluate with a for the preparation of effective biocatalysts to be employed
purification factor of 160 [58]. It is noteworthy that the both in aqueous and water-poor media. The release of
combination of integrated product isolation from whole cell affinity-bound cells from the elastic cryogels under mild
suspensions using macroporous cryogels with the specific conditions (i.e. via a mechanical compression of the cryogel)
format of cryogel columns (i.e. cryogels in a 96-well plate) ensures a high viability and recovery of the desorbed cells
allowed for the construction of an efficient 96-cryogel plat- during cell chromatography. Furthermore, the large size of
form for high throughput screening of complex biological interconnected pores in cryogel monoliths permits a fast
samples. processing of considerable volumes of liquid phase. Thus,
the macroporous cryogel monolithic filters, bearing specific
Concluding remarks and future perspectives ligands, can be used for the analytical detection or selective
The unique combination of properties of macroporous capture of specific cells in water effluents. The use of
cryogels, such as their controlled macroporous structure, cryogels can be envisaged for analyzing the composition

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