Questions and Answers

Start ] About us ] References ] Products ] Quality Control ] [ Questions and Answers ] Microcarriers ] Application Notes ] Picture Gallery ]

 

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.

 

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.

 

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.

 

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.

 

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.

 

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.

 

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.

 

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.

 

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.

 

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).

 

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.

 

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.

 

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.

 

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.

 

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.

 

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

 

BSE CultiSpher is based on porcine skin gelatin which is considered as free from BSE. Further details in quality control section.