A collection of commonly asked questions and their answers. Help us to improve this list by submitting your specific questions.
| Storage and stability | CultiSpher is supplied as a dry
powder and is hydrated and sterilized before use. Packs should be stored under
dry conditions at room temperature. Expiration date is marked on the package.
CultiSpher has an expiration time of 4 years from production.
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| Scale-up | Cultures of cells grown on CultiSpher can be
scaled-up in steps of 50 times; for instance the cells harvested from a 1 l
fermentor are sufficient to inoculate a 50 litres fermentor. This is possible
due to the macroporous structure and the digestible gelatin matrix. The
macroporous structure allows the cells to increase from 10-20 to 2000-3000 on
each bead. As the matrix is easily digested with tissue culture grade trypsin
all these cells can be recovered and used for scale-up purposes.
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| Sampling and cell counting | Mix the culture and make sure
that the microcarriers are evenly dispersed. Take duplicate samples of 0.5 ml.
After sedimentation of beads, 0.3 ml of the supernatant is removed. Add 0.8 ml
trypsin(0.25 %, w/v in PBS). Mix and incubate at 37 °C for 20 minutes. If the
microcarriers have not dissolved after this time increase trypsin concentration.
Disperse the cells with a Pasteur pipette and count the cells directly or after
viability staining with Trypan Blue.
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| Microcarrier concentration | Use 1-2 g/l. Used at 1 g/l cell concentrations of 10 x 106
cells/ml can be obtained if very efficient oxygen supply system are used.
If used in higher concentrations than 2 g/l in standard systems, cells will
only grow in the outer layer of the microcarriers. Growth is dependent on the
oxygen gradient within the system. Cells will only grow as long as they are provided with
sufficent amounts of oxygen. Remember that pH is usually controlled by
carbon dioxide, if a large number of cells is growing inside the beads
they will produce an acidic environment. Unless this is controlled it will
affect both cell growth and product formation.
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| Which type to use | Start by testing CultiSpher-S. This type
has a faster rate of cell attachment. It can be autoclaved for extended times.
1-2 hours. It will take longer time, 15-45 minutes, to be dissolved for cell
harvesting.
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| Starting volume | Cell attachment is slower than to charged microcarriers. Use as small volume as possible as this increases the possibility
of cell-microcarrier collision. Adjust the volume to final volume on the second or
third day.
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| Starting cell number | Example: a 1 l fermentor inoculated
with the smallest possible volume. CultiSpher used at 2 g/l contains 2 x 106
beads. Each bead should at least be inoculated with 10 cells. Required number of
cells = 20 x 106. Normally cells must be used above a minimum
concentration for optimal attachment and growth. If this concentration is
0.1 x 106cells/ml and the minimum volume that can be used is 0.5 l,
the necessary amount of cells will be 50 x 106. The culture must therefore
be started with 50 x 106 cells which will supply each
CultiSpher bead with 25 cells.
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| Serum-free conditions | Cell adhesion is mediated by the
protein fibronektin. Fibronektin is present in serum and will attach to the
gelatin microcarriers through an affinity interaction (this is a method to purify
fibronektin). In serum-free culture it is necessary to either add fibronektin or precoat the
microcarriers by incubation with a serum-containing
medium. After a growth period in a serum-containing medium a serum-free medium
can be used for a prolonged production period. Cells will continue to adhere
firmly during this period.
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| Agitation rate | Use as slow agitation as possible in the
first 24 hours, just enough to keep the microcarriers suspended. The majority of
cells will attach during the first 24 hours. Usually no increase in cell
concentrations will be noticed during this time. The agitation rate is increased
during a later phase of growth to improve oxygenation and prevent aggregation
of microcarriers.
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| Electron microscopy | Wash the beads twice with PBS. Add PBS
with glutardialdehyde(4%, v/v) and incubate at 4 °C overnight. Dehydrate in a
graded series of ethanol solutions. Dry in vacuum. Transfer to SEM equipment and
sputter-coat with gold. An improved procedure is described in Microscopy Res.
Techn. 30, 419-426 (1995).
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| MTT- staining | MTT (3-(4,5-dimethylthiazol-2-yl)-2,
5-diphenyltetrazolium bromide) is cleaved by an enzyme in the respiration chain
in the mitochondria if the cell is viable, generating MTT formazan a dark blue, highly visible
product. Wash the microcarriers with PBS. Add a solution of
MTT(5 mg/ml in PBS, 1 part MTT solution to 10 parts microcarriers). Incubate for
45 minutes at 37 °C. Observe the cells with a light or phase-contrast
microscope.
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| Cell harvest | Let the microcarriers sediment for 10 minutes.
Remove the supernatant. Wash twice with PBS-EDTA(0.02%, w/w EDTA), 50 ml/g dry
CultiSpher. Replace the PBS-EDTA with trypsin solution(0.1% in PBS containing Ca2+
and Mg2+, pH 8.0. Incubate at 37 °C for 15 to 30 minutes with
occasional agitation. It is important to adjust the concentration of trypsin so
that complete dissolution of the beads are obtained within the designated time. A
viability of 95-100 % should be obtained.
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| Cell yields | Expect cell yields -3 times greater compared to a
solid matrix microcarrier. This is highly dependent on the type of culture systems used.
Normally cultures containing cell concentrations above 2- 3 x 106 cells/ml will be more or less
depleted of oxygen in a short time. The result is growth of cells in only in the outer region
of the macroporous structure. Unless active oxygen supply is employed
microcarrier concentrations above 2 g/l will yield poor results. Consult Application
Note 100 for further information.
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| Primary cells | CultiSpher microcarriers are made of gelatin.
Gelatin is a product derived from the partial hydrolysis of collagen and have large regions
of identical structure. The
majority of primary cells will attach to the gelatin structure. Since the
macroporous microcarrier structure creates a three-dimensional framework it
mimics the cooperative conformation demonstrated in tissue.
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| Density | The density is around 1.02 - 1.04 g/ml. When the
beads are filled with cells their weight will significantly increase the density.
This is one of the reasons for increasing agitation rate during later parts of
the culture.
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| Clumping of microcarriers | This is a general problem in
microcarrier cell culture. We recommend an article in Cytotechnology 8:237,
237-248 1992: Formation of bridges and large cellular clumps in CHO-cells
microcarrier cultures: effects of agitation, dimethylsulfoxide and calf serum.
Normally this problem is best solved by a gradual increase in agitation rate
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| BSE | CultiSpher is based on porcine skin gelatin which is
considered as free from BSE. Further details in quality
control section.
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