Seplife® LX-MC-Dex1

Guidelines of the instructions for use are presented below. The user should perform process optimization at all stages in the Seplife®LX-MC-Dex1 usage.

3.1 Microcarrier pretreatment

Rehydrate the dried microcarriers ( 3-5g microcarrier/L culture volume is recommended) with Ca²⁺ and Mg²⁺ free phosphate saline buffer, pH=7.4 (at least 80ml/g microcarriers) at room temperature for at least 3 hours.

Discard the supernatant and rinse the microcarriers with freshly prepared Ca²⁺ and Mg²⁺ free phosphate buffer, pH=7.4 for 2-3 times.

Sterilize the microcarrier solution by autoclaving (121°C, 30min, 15psi).

The microcarrier Seplife®LX-MC-Dex1 is very stable and can be autoclaved repeatedly (at least 5 times) or for a long time (130°C, 12h, 27psi) without impact on its performance.

After sterilization, discard the supernatant and rinse the beads with culture medium for 2-3 times. Pretreatment with complete culture medium can be done if needed.

3.2 Microcarrier culture

Add the digested cells into prepared microcarrier culture system and start culturing under controlled conditions.

Observe the cell attachment after 3-8 hours. If the cells are well attached, continue culturing and monitor the growth of the cells.

3.3 Key points of microcarrier culture operation

- Attach period: ensure that the culture medium and microcarrier are at a stable pH and temperature level. Small initial culture volume such as 1/3 of the final volume should be applied to improve the attachment of the cells on the surface of microcarriers. Higher microcarrier concentrations could be used as well, which requires highly-controlled culture system or more frequent culture medium exchange. Lower stirring speed or interval stirring is preferred during this period for better attachment.

After attachment (3-8h), slowly add the culture medium to the final culture volume, and increase the stirring speed to ensure proper and homogeneous mixing.

- Culture period: Cell counts and morphology microscopy are usually used to evaluate the culture performance. As the cells proliferate, the beads become heavier and the stirring speed might need to be increased. During the culture process, parameters such as pH, temperature, dissolved oxygen, should be monitored to keep the culture under optimized condition. Usually, phenol red is used as an indicator for lab scale culture for medium exchange, pH adjustment or/and nutrients supplement.

- Cell harvest: Discard the culture medium and rinse with Ca²⁺ and Mg²⁺ free phosphate saline buffer, pH=7.4 sterile at least once. Add dissociation enzyme such as trypsin to detach the cells from the surface of microcarriers, then use serum or other reagent to terminate digestion. Use suitable cell strainer to separate cells from microcarrier beads. If the molecule of interest is secreted by cells in the medium or the virus of interest is released from cells, a proper filtration should be applied to separate microcarriers with cells from culture medium.

- Scale-up of microcarrier cell culture: large scale microcarrier cell culture can be achieved from small scale culture step by step through increasing of the culture volume with the same microcarrier concentration. 

Store in closed containers at 4-30℃, in a dry, ventilated and clean place, away from direct sunlight.